Mammalian Cytokines; Related Reagents and Methods

ABSTRACT

Nucleic acids encoding mammalian, e.g., primate, IL-ζ, purified IL-1ζ polypeptides and fragments thereof. Binding proteins, e.g., antibodies, both polyclonal and monoclonal, are also provided. Methods of using the compositions for both diagnostic and therapeutic utilities are provided.

This filing is a conversion from U.S. Provisional Patent ApplicationU.S. Ser. No. 60/100,948, filed Sep. 18, 1998, to a U.S. Utility PatentApplication.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for affectingmammalian physiology, including morphogenesis or immune system function.In particular, it provides nucleic acids, proteins, and antibodies whichregulate development and/or the immune system. Diagnostic andtherapeutic uses of these materials are also disclosed.

BACKGROUND OF THE INVENTION

Recombinant DNA technology refers generally to techniques of integratinggenetic information from a donor source into vectors for subsequentprocessing, such as through introduction into a host, whereby thetransferred genetic information is copied and/or expressed in the newenvironment. Commonly, the genetic information exists in the form ofcomplementary DNA (cDNA) derived from messenger RNA (mRNA) coding for adesired protein product. The carrier is frequently a plasmid having thecapacity to incorporate cDNA for later replication in a host and, insome cases, actually to control expression of the cDNA and therebydirect synthesis of the encoded product in the host.

For some time, it has been known that the mammalian immune response isbased on a series of complex cellular interactions, called the “immunenetwork”. Recent research has provided new insights into the innerworkings of this network. While it remains clear that much of the immuneresponse does, in fact, revolve around the network-like interactions oflymphocytes, macrophages, granulocytes, and other cells, immunologistsnow generally hold the opinion that soluble proteins, known aslymphokines, cytokines, or monokines, play critical roles in controllingthese cellular interactions. Thus, there is considerable interest in theisolation, characterization, and mechanisms of action of cell modulatoryfactors, an understanding of which will lead to significant advancementsin the diagnosis and therapy of numerous medical abnormalities, e.g.,immune system disorders.

Lymphokines apparently mediate cellular activities in a variety of ways.They have been shown to support the proliferation, growth, and/ordifferentiation of pluripotent hematopoietic stem cells into vastnumbers of progenitors comprising diverse cellular lineages which makeup a complex immune system. Proper and balanced interactions between thecellular components are necessary for a healthy immune response. Thedifferent cellular lineages often respond in a different manner whenlymphokines are administered in conjunction with other agents.

Cell lineages especially important to the immune response include twoclasses of lymphocytes: B-cells, which can produce and secreteimmunoglobulins (proteins with the capability of recognizing and bindingto foreign matter to effect its removal), and T-cells of various subsetsthat secrete lymphokines and induce or suppress the B-cells and variousother cells (including other T-cells) making up the immune network.These lymphocytes interact with many other cell types.

Another important cell lineage is the mast cell (which has not beenpositively identified in all mammalian species), which is agranule-containing connective tissue cell located proximal tocapillaries throughout the body. These cells are found in especiallyhigh concentrations in the lungs, skin, and gastrointestinal andgenitourinary tracts. Mast cells play a central role in allergy-relateddisorders, particularly anaphylaxis as follows: when selected antigenscrosslink one class of immunoglobulins bound to receptors on the mastcell surface, the mast cell degranulates and releases mediators, e.g.,histamine, serotonin, heparin, and prostaglandins, which cause allergicreactions, e.g., anaphylaxis.

Research to better understand and treat various immune disorders hasbeen hampered by the general inability to maintain cells of the immunesystem in vitro. Immunologists have discovered that culturing many ofthese cells can be accomplished through the use of T-cell and other cellsupernatants, which contain various growth factors, including many ofthe lymphokines.

The interleukin-1 family of proteins includes the IL-1_, the IL-1_, theIL-1RA, and recently the IL-1_(—) (also designated Interferon-GammaInducing Factor, IGIF). This related family of genes has been implicatedin a broad range of biological functions. See Dinarello (1994) FASEB J.8:1314-1325; Dinarello (1991) Blood 77:1627-1652; and Okamura, et al.(1995) Nature 378:88-91.

In addition, various growth and regulatory factors exist which modulatemorphogenetic development. This includes, e.g., the Toll ligands, whichsignal through binding to receptors which share structural, andmechanistic, features characteristic of the IL-1 receptors. See, e.g.,Lemaitre, et al. (1996) Cell 86:973-983; and Belvin and Anderson (1996)Ann. Rev. Cell & Develop. Biol. 12:393-416.

From the foregoing, it is evident that the discovery and development ofnew soluble proteins, including ones similar to lymphokines, shouldcontribute to new therapies for a wide range of degenerative or abnormalconditions which directly or indirectly involve development,differentiation, or function, e.g., of the immune system and/orhematopoietic cells. In particular, the discovery and understanding ofnovel lymphokine-like molecules which enhance or potentiate thebeneficial activities of other lymphokines would be highly advantageous.The present invention provides new interleukin-1 like compositions andrelated compounds, and methods for their use.

SUMMARY OF THE INVENTION

The present invention is based on the discovery, purification, andcharacterization of the biological activities of a novel mammalian,e.g., primate, interleukin-1 like molecule, designated interleukin-1ζ(IL-1ζ). IL-1ζ exhibits both structural and sequence similarity, e.g.,by homology comparison, to known members of the IL-1 family ofmolecules.

In a first aspect, the invention provides an isolated or recombinantpolypeptide that: specifically binds polyclonal antibodies generatedagainst at least a 12 consecutive amino acid segment of SEQ ID NO: 2 or4; and comprises at least one sequence selected from: GENSGVK; EDWEKD;CCLEDPA; FVHTSR; KKFSIHD; VLVLDS; NLIAVP; FFALAS; SSASAEK; SLILLGV;FCLYCDK; PSLQLK; KLMKLAAQ; FIFYRAQ; SRNMLES; WFICTS; EPVGVT; or FSFQPVC(see SEQ ID NO: 2); or FVHTSP; SPILLGV; or SWNMLES (see SEQ ID NO: 4).Certain embodiments include those: wherein the polypeptide comprises aplurality of the sequence; or which specifically bind to polyclonalantibodies generated against an immunogen selected from SEQ ID NO: 2 or4. Other embodiments include those where the 12 consecutive amino acidsegment is selected from: GVKMGSEDWEKD; AGSPLEPGPSLP; SRKVKSLNPKKF;HDQDHKVLVLDS; NLIAVPDKNYIR; FALASSLSSASA; GQSHPSLQLKKE; MKLAAQKESARR;FYRAQVGSRNML; TSCNCNEPVGVT; FENRKHIEFSFQ; or PVCKAEMSPSEV (see SEQ IDNO: 2); or AVSPLEPGPSLP; SPKVKNLNPKKF; or FYRAQVGSWNML (see SEQ ID NO:4). Certain preferred embodiments include those wherein the polypeptide:comprises a mature protein; lacks a post-translational modification; isfrom a primate, including a human; is a natural allelic variant ofIL-1ζ; has a length at least about 30 amino acids; exhibits at least twonon-overlapping epitopes that are specific for a primate IL-1ζ; exhibitsa sequence identity over a length of at least about 20 amino acids toSEQ ID NO: 2 or 4; is not glycosylated; has a molecular weight of atleast 10 kD with natural glycosylation; is a synthetic polypeptide; isattached to a solid substrate; is conjugated to another chemical moiety;is a 5-fold or less substitution from natural sequence; or is a deletionor insertion variant from a natural sequence.

Other embodiments include a soluble polypeptide comprising: the sterilepolypeptide; the sterile polypeptide and a carrier, wherein the carrieris: an aqueous compound, including water, saline, and/or buffer; and/orformulated for oral, rectal, nasal, topical, or parenteraladministration.

Fusion protein embodiments include those having a polypeptide sequencedescribed, further comprising: a mature polypeptide as described; adetection or purification tag, including a FLAG, His6, or Ig sequence;or sequence of another cytokine or chemokine.

Kit embodiments include those comprising such a polypeptide and: acompartment comprising said polypeptide; and/or instructions for use ordisposal of reagents in said kit.

Antibody or binding compound embodiments encompass a binding compoundcomprising an antigen binding site from an antibody, which specificallybinds to a mature polypeptide, as described, wherein: the maturepolypeptide is a primate IL-1ζ; the binding compound is an Fv, Fab, orFab2 fragment; the binding compound is conjugated to another chemicalmoiety; or the antibody: is raised against a 12 consecutive amino acidsegment of SEQ ID NO: 2 or 4; is raised against a mature IL-1ζ; israised to a purified primate IL-1ζ; is immunoselected; is a polyclonalantibody; binds to a denatured IL-1ζ; exhibits a Kd to antigen of atleast 30 μM; is attached to a solid substrate, including a bead orplastic membrane; is in a sterile composition; or is detectably labeled,including a radioactive or fluorescent label. An alternative bindingcompound embraces one comprising an antigen binding portion from anantibody, which specifically binds to a primate protein, as described,wherein: the protein is a human protein; the binding compound is an Fv,Fab, or Fab2 fragment; the binding compound is conjugated to anotherchemical moiety; or the antibody: is raised against a polypeptidesequence of a mature polypeptide comprising at least 12 consecutiveamino acids of SEQ ID NO: 2 or 4; is raised against a mature primateIL-1ζ; is raised to a purified primate IL-1ζ; is immunoselected; is apolyclonal antibody; binds to a denatured primate IL-1ζ; exhibits a Kdto antigen of at least 30 μM; is attached to a solid substrate,including a bead or plastic membrane; is in a sterile composition; or isdetectably labeled, including a radioactive or fluorescent label. Kitsare provided comprising the binding compound, as described, and: acompartment comprising said binding compound; and/or instructions foruse or disposal of reagents in the kit. Methods are also provided, e.g.,of: making an antibody, comprising immunizing an immune system with animmunogenic amount of: a primate IL-1ζ polypeptide; or a peptidesequence comprising at least 12 consecutive amino acids of SEQ ID NO: 2or 4; thereby causing said antibody to be produced; or producing anantigen:antibody complex, comprising contacting a primate IL-1ζpolypeptide with an antibody, as described, thereby allowing saidcomplex to form.

The invention further embraces a composition comprising: the sterilebinding compound described, or the binding compound and a carrier,wherein the carrier is: an aqueous compound, including water, saline,and/or buffer; and/or formulated for oral, rectal, nasal, topical, orparenteral administration.

Nucleic acid embodiments include an isolated or recombinant nucleic acidencoding the described polypeptide, wherein: the polypeptide is aprimate IL-1ζ; or the nucleic acid: encodes an antigenic peptidesequence of SEQ ID NO: 2 or 4; encodes a plurality of antigenic peptidesequences of SEQ ID NO: 2 or 4; exhibits at least about 80% identity toa natural cDNA encoding said segment; is an expression vector; furthercomprises an origin of replication; is from a natural source; comprisesa detectable label; comprises synthetic nucleotide sequence; is lessthan 6 kb, preferably less than 3 kb; is from a rodent; comprises anatural full length coding sequence; is a hybridization probe for a geneencoding said IL-1ζ; or is a PCR primer, PCR product, or mutagenesisprimer; or encodes an IL-1ζ polypeptide. The invention also provides acell transformed with the described nucleic acid, e.g., where the cellis: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeastcell; an insect cell; a mammalian cell; a mouse cell; a primate cell; ora human cell.

Another embodiment is a kit comprising the described nucleic acid and: acompartment comprising said nucleic acid and: a compartment furthercomprising a primate IL-1ζ polypeptide; and/or instructions for use ordisposal of reagents in said kit.

Other nucleic acid embodiments include an isolated or recombinantnucleic acid that hybridizes under wash conditions of 30° C. and lessthan 2M salt to SEQ ID NO: 1; or where the wash condition is at: 45° C.and/or 500 mM salt; 55° C. and/or 150 mM salt; or encodes at least 12 or17 contiguous amino acids of SEQ ID NO: 2 or 4.

The invention also provides methods of modulating a cell involved in aninflammatory response comprising contacting said cell with an agonist orantagonist of a primate IL-1ζ polypeptide. Preferably, the contacting isin combination with an agonist or antagonist of IL-1α, IL-1RA, IL-1β,IL-1γ, IL-1δ, IL-1ε, IL-2, and/or IL-12; the contacting is with anantagonist, including binding composition comprising an antibody bindingsite which specifically binds an IL-1ζ; or the modulating is regulationof IFN-γ production.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Outline

I. General II. Activities

III. Nucleic acids

A. encoding fragments, sequence, probes

B. mutations, chimeras, fusions

C. making nucleic acids

D. vectors, cells comprising

IV. Proteins, Peptides

A. fragments, sequence, immunogens, antigens

B. muteins

C. agonists/antagonists, functional equivalents

D. making proteins

V. Making nucleic acids, proteins

VI. Antibodies

A. polyclonals

B. monoclonal, Kd

C. anti-idiotypic antibodies

D. hybridoma cell lines

VII. Kits and Methods to quantify IL-1ζ

A. ELISA

B. assay mRNA encoding

C. qualitative/quantitative

D. kits

VIII. Therapeutic compositions, methods

A. combination compositions

B. unit dose

C. administration

IX. Receptors I. General

Before the present compositions, formulations, and methods aredescribed, it is to be understood that this invention is not limited tothe particular methods, compositions, and cell lines described herein,as such methods, compositions, and cell lines may, of course, vary. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which is only defined by theappended claims.

As used herein, including the appended claims, singular forms of wordssuch as “a,” “an,” and “the” include their corresponding pluralreferents unless the context clearly dictates otherwise. Thus, e.g.,reference to “an organism” includes one or more different organisms,reference to “a cell” includes one or more of such cells, and referenceto “a method” includes reference to equivalent steps and methods knownto a person of ordinary skill in the art, and so forth.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references discussed above are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of its priorinvention. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety including all figures and drawings.

The present invention provides the amino acid sequence and DNA sequenceof a mammalian, e.g., human, interleukin-1 like molecule havingparticular defined properties, both structural and biological. This hasbeen designated herein as interleukin-1ζ, and increases the number ofmembers of the IL-1 family from 6 to 7. Various cDNAs encoding thesemolecules were obtained from primate, e.g., human, cDNA sequencelibraries. Rodent counterparts should also exist. The nucleic acidsencompassed herein include DNA, cDNA, and RNA sequences which encodeIL-1ζ. It is understood that nucleic acids encoding all or a portion ofIL-1ζ polypeptides are also encompassed, so long as they encode apolypeptide with IL-1ζ activity. Such nucleic acids include bothnaturally occurring and intentionally manipulated nucleic acids. Forexample, IL-1ζ may be subjected to site-directed mutagenesis.

Some of the standard methods applicable are described or referenced,e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, etal. (1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols 1-3,CSH Press, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and periodic supplements)Current Protocols in Molecular Biology, Greene/Wiley, New York; each ofwhich is incorporated herein by reference.

A complete nucleotide (SEQ ID NO: 1) and corresponding amino acidsequence (SEQ ID NO: 2) of a primate IL-1ζ coding segment is shown inTable 1. An alternative sequence, perhaps an allelic variant, ispresented as SEQ ID NO: 3 and 4. Table 2 provides an alignment ofselected family members.

TABLE 1 Nucleotide and amino acid sequences (see SEQ ID NO: 1-4) ofmammalian, e.g., primate, IL-1ξ. The coding sequence does notindicate a signal sequence, which has been reported forvarious forms of messages encoding other members of theIL-1 family. It is likely that another form of the messageprobably encodes a signal sequence, much like the IL-1βprodomain which is cleaved by a convertase-like enzyme, see Dinarello (1994) FASEB J. 1314-1325).CGGTTTGTTT TCTTTAGAGA TTTTACAGTG TTGGTTATAA TTGTGCACTT AATCTTTATT 60TTCCTTATAC AGTAGTCCCC CCGATCAACT GGGGGCATGT TCCATACCCC TGGTGGATTC 120CTGAAACTGC CAGTTAGTAC CAAACCCTAT ATAGATTGTG TTTTTTCCTG TACGCAGGCC 180GACACACAGG AAATCATAAG TCAGGAGGGC CACTGCCACG CAGGAAAGAC CCATCTGAAC 240TGCTGCAAAA GCTCCGTGTC GATTTATTGC TTCCACAAAT AGTGCCGATA TGCACCAGGC 300ACTGTTGTAA AACTGAAAAT ATGTTTTGGG ATGTGCCCAG TCTACCTAGC TCCTTCAAGT 360AAAGGATCCT GAGAACTGAA GGCAAACAGA GCTCCAGGAG TCCAAGACAG AGCCACACAC 420CACGAGGATC CTGGCCCAGG TCTTGGACTT CCATTCCCAT TTCTGTTGAG TAATAAACTC 480AACGTTGAAA ATG TCC TTT GTG GGG GAG AAC TCA GGA GTG AAA ATG GGC 529           Met Ser Phe Val Gly Glu Asn Ser Gly Val Lys Met Gly             1               5                  10TCT GAG GAC TGG GAA AAA GAT GAA CCC CAG TGC TGC TTA GAA GAC CCG 577Ser Glu Asp Trp Glu Lys Asp Glu Pro Gln Cys Cys Leu Glu Asp Pro     15                  20                  25GCT GGA AGC CCC CTG GAA CCA GGC CCA AGC CTC CCC ACC ATG AAT TTT 625Ala Gly Ser Pro Leu Glu Pro Gly Pro Ser Leu Pro Thr Met Asn Phe 30                  35                  40                  45GTT CAC ACA AGT CGA AAG GTG AAG AGC TTA AAC CCG AAG AAA TTC AGC 673Val His Thr Ser Arg Lys Val Lys Ser Leu Asn Pro Lys Lys Phe Ser                 50                  55                  60ATT CAT GAC CAG GAT CAC AAA GTA CTG GTC CTG GAC TCT GGG AAT CTC 721Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu             65                  70                  75ATA GCA GTT CCA GAT AAA AAC TAC ATA CGC CCA GAG ATC TTC TTT GCA 769Ile Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala         80                  85                  90TTA GCC TCA TCC TTG AGC TCA GCC TCT GCG GAG AAA GGA AGT CTG ATT 817Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Leu Ile     95                 100                 105CTC CTG GGG GTC TCT AAA GGG GAG TTT TGT CTC TAC TGT GAC AAG GAT 865Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp110                 115                 120                 125AAA GGA CAA AGT CAT CCA TCC CTT CAG CTG AAG AAG GAG AAA CTG ATG 913Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met                130                 135                 140AAG CTG GCT GCC CAA AAG GAA TCA GCA CGC CGG CCC TTC ATC TTT TAT 961Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr            145                 150                 155AGG GCT CAG GTG GGC TCC CGG AAC ATG CTG GAG TCG GCG GCT CAC CCC 1009Arg Ala Gln Val Gly Ser Arg Asn Met Leu Glu Ser Ala Ala His Pro        160                 165                 170GGA TGG TTC ATC TGC ACC TCC TGC AAT TGT AAT GAG CCT GTT GGG GTG 1057Gly Trp Phe Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val    175                 180                 185ACA GAT AAA TTT GAG AAC AGG AAA CAC ATT GAA TTT TCA TTT CAA CCA 1105Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro190                 195                 200                 205GTT TGC AAA GCT GAA ATG AGC CCC AGT GAG GTC AGC GAT TAGGAAACTG 1154Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp                210                 215CCCCATTGAA CGCCTTCCTC GCTAATTTGA ACTAATTGTA TAAAAACCCC AAACCTGCTC 1214ACTAAAAAAA A 1225MSFVGENSGVKMGSEDWEKDEPQCCLEDPAGSPLEPGPSLPTMNFVHTSRKVKSLNPKKFSIHDQDHKVLVLDSGNLIAVPDKNYIRPEIFFALASSLSSASAEKGSLILLGVSKGEFCLYCDKDKGQSHPSLQLKKEKLMKLAAQKESARRPFIFYRAQVGSRNMLESAAHPGWFICTSCNCNEPVGVTDKFENRKHIEFSFQPVCKAEMSPSEVSDSequence of a second variant form, which exhibits nucleotide basechanges at nucleotide positions of the coding region: 92 (G -> T;changing amino acids G -> V); 124 (A -> G; changing T -> A); 149 (G ->C; changing R -> P); 161 (G -> A; changing S -> N);  323 (T ->C; changing L -> P); and 490 (C -> T; changing R -> W).ATG TCC TTT GTG GGG GAG AAC TCA GGA GTG AAA ATG GGC TCT GAG GAC 48Met Ser Phe Val Gly Glu Asn Ser Gly Val Lys Met Gly Ser Glu Asp  1               5                  10                  15TGG GAA AAA GAT GAA CCC CAG TGC TGC TTA GAA GAC CCG GCT GTA AGC 96Trp Glu Lys Asp Glu Pro Gln Cys Cys Leu Glu Asp Pro Ala Val Ser             20                  25                  30CCC CTG GAA CCA GGC CCA AGC CTC CCC GCC ATG AAT TTT GTT CAC ACA 144Pro Leu Glu Pro Gly Pro Ser Leu Pro Ala Met Asn Phe Val His Thr         35                  40                  45AGT CCA AAG GTG AAG AAC TTA AAC CCG AAG AAA TTC AGC ATT CAT GAC 192Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser Ile His Asp     50                  55                  60CAG GAT CAC AAA GTA CTG GTC CTG GAC TCT GGG AAT CTC ATA GCA GTT 240Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu Ile Ala Val 65                  70                  75                  80CCA GAT AAA AAC TAC ATA CGC CCA GAG ATC TTC TTT GCA TTA GCC TCA 288Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala Leu Ala Ser                 85                  90                  95TCC TTG AGC TCA GCC TCT GCG GAG AAA GGA AGT CCG ATT CTC CTG GGG 336Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile Leu Leu Gly            100                 105                 110GTC TCT AAA GGG GAG TTT TGT CTC TAC TGT GAC AAG GAT AAA GGA CAA 384Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp Lys Gly Gln        115                 120                 125AGT CAT CCA TCC CTT CAG CTG AAG AAG GAG AAA CTG ATG AAG CTG GCT 432Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met Lys Leu Ala    130                 135                 140GCC CAA AAG GAA TCA GCA CGC CGG CCC TTC ATC TTT TAT AGG GCT CAG 480Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr Arg Ala Gln145                 150                 155                 160GTG GGC TCC TGG AAC ATG CTG GAG TCG GCG GCT CAC CCC GGA TGG TTC 528Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro Gly Trp Phe                165                 170                 175ATC TGC ACC TCC TGC AAT TGT AAT GAG CCT GTT GGG GTG ACA GAT AAA 576Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val Thr Asp Lys            180                 185                 190TTT GAG AAC AGG AAA CAC ATT GAA TTT TCA TTT CAA CCA GTT TGC AAA 624Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro Val Cys Lys        195                 200                 205GCT GAA ATG AGC CCC AGT GAG GTC AGC GAT TAG 657Ala Glu Met Ser Pro Ser Glu Val Ser Asp     210                 215MSFVGENSGVKMGSEDWEKDEPQCCLEDPAVSPLEPGPSLPAMNFVHTSPKVKNLNPKKFSIHDQDHKVLVLDSGNLIAVPDKNYIRPEIFFALASSLSSASAEKGSPILLGVSKGEFCLYCDKDKGQSHPSLQLKKEKLMKLAAQKESARRPFIFYRAQVGSWNMLESAAHPGWFICTSCNCNEPVGVTDKFENRKHIEFSFQPVCKAEMSPSEVSD

TABLE 2 IL-1a_human.......... .......... .......... ........SA PFSFLSNVKY IL-1a_mouse.......... .......... .......... ........SA PYTYQSDLRY IL-1g_human.......... .......... .......... .......... .......... IL-1g_mouse.......... .......... .......... .......... .......... IL-1b_human.......... .......... .......... .......... .......... IL-1b_mouse.......... .......... .......... .......... .......... IL-1x_human.......... .......... .......... .......... ......CRPS IL-1x_mouse.......... .......... .......... .......... ......CRPS IL-1d_mouse.......... .......... .......... .......... .......... IL-1z_humanMSFVGENSGV KMGSEDWEKD EPQCCLEDPA GSPLEPGPSL PTMNFVHTSR IL-1e_mouse.......... .......... .......... .......... .....MNKEK IL-1e_human.......... .......... .......... .....MRGTP GDADGGGRAV IL-1a_humanNFMRIIKYEF ILNDALN... QSIIRAND.. QYLTAAALHN LD...EAVKF IL-1a_mouseKLMKLVRQKF VMNDSLN... QTIYQDVD.K HYLSTTWLND LQ...QEVKF IL-1g_human..DYFGKLES KLSVIRNLND QVLFIDQGNR PLFEDMTDSD CRDNAPRTIF IL-1g_mouse..DNFGRLHC TTAVIRNIND QVLFVDKR.Q PVFEDMTDID QSASEPQTRL IL-1b_human.DAPVRSLNC TLRDSQQ... KSLVMSGP.. YELKALHLQG QDM.EQQVVF IL-1b_mouse.DVPIRQLHY RLRDEQQ... KSLVLSDP.. YELKALHLNG QNI.NQQVIF IL-1x_humanGRKSSKMQAF RIWDVNQ... KTFYLRN... NQLVAGYLQG PNV.NLEEKI IL-1x_mouseGKRPCKMQAF RIWDTNQ... KTFYLRN... NQLIAGYLQG PNI.KLEEKI IL-1d_mouseMMVLSGALCF RMKDSAL... KVLYLHN... NQLLAGGLHA EKVIKGEEIS IL-1z_humanKVKSLNPKKF SIHDQDH... KVLVLDS... GNLIAVPDKN YIR..PEIFF IL-1e_mouseELRAASPSLR HVQDLSS... RVWILQN... NILTAVPRKE QTV..PVTIT IL-1e_humanYQSMCKPITG TINDLNQ... QVWTLQG... QNLVAVPRSD SVT..PVTVA IL-1a_humanDMGAYKSSK. .DDAKITVIL RISK.TQLYV TAQD....ED QPVLLKEMPE IL-1a_mouseDMYAYSSGG. .DDSKYPVTL KISD.SQLFV SAQG....ED QPVLLKELPE IL-1g_humanIISMYKDS.. .QPRGMAVTI SVKCEKISTL SCEN...... KIISFKEMNP IL-1g_mouseIIYMYKDS.. .EVRGLAVTL SVKDSKMSTL SCKN...... KIISFEEMDP IL-1b_humanSMSFVQGEE. .SNDKIPVAL GLKE.KNLYL SCVL.KD.DK PTLQLESVDP IL-1b_mouseSMSFVQGEP. .SNDKIPVAL GLKG.KNLYL SCVM.KD.GT PTLQLESVDP IL-1x_humanDVVPIEP... .....HALFL GIHG.GKLCL SCVK.SG.DE TRLQLEAVNI IL-1x_mouseDMVPIDL... .....HSVFL GIKG.YKLYM SCVK.SG.DD IKLQLEEVNI IL-1d_mouseVVPNRALD.. ..ASLSPVIL GVQG.GSQCL SCGT..E.KG PILKLEPVNI IL-1z_humanALASSLSSAS .AEKGSLILL GVSK.GEFCL YCDKDKGQSH PSLQLKKEKL IL-1e_mouseLLPCQYLDTL ETNRGDPTYM GVQR.PMSCL FCTK..DGEQ PVLQLGEGNI IL-1e_humanVITCKYPEAL EQGRGDPIYL GIQN.PEMCL YCEK..VGEQ PTLQLKEQKI IL-1a_humanIPKTITG..S ETNLLFFWET HG...TKNYF TSVAHPNLFI ATKQ...DYW IL-1a_mouseTPKLITG..S ETDLIFFWKS IN...SKNYF TSAAYPELFI ATKE...QSR IL-1g_humanPDNIKD...T KSDIIFFQRS VPGHDNKMQF ESSSYEGYFL ACEKERDLFK IL-1g_mousePENIDD...I QSDLIFFQKR VPGH.NKMEF ESSLYEGHFL ACQKEDDAFK IL-1b_humanKNYPKK..KM EKRFVFNKIE IN...NKLEF ESAQFPNWYI STSQA.ENMP IL-1b_mouseKQYPKK..KM EKRFVFNKIE VK...SKVEF ESAEFPNWYI STSQA.EHKP IL-1x_humanTDLSENR.KQ DKRFAFIRSD SG...PTTSF ESAACPGWFL CTAME.ADQP IL-1x_mouseTDLSKNK.EE DKRFTFIRSE KG...PTTSF ESAACPGWFL CTTLE.ADRP IL-1d_mouseMELYLGA.KE SKSFTFYRRD MG...LTSSF ESAAYPGWFL CTSPE.ADQP IL-1z_humanMKLAAQKESA RRPFIFYRAQ VG...SRNML ESAAHPGWFI CTSCN.CNEP IL-1e_mouseMEMYNKK.EP VKASLFYHKK SG...TTSTF ESAAFPGWFI AVCSK.GSCP IL-1e_humanMDLYGQP.EP VKPFLFYRAK TG...RTSTL ESVAFPDWFI ASSKR..DQP IL-1a_humanVCLAG..... .GPPSITDFQ ILENQA.... ...... IL-1a_mouseVHLAR..... .GLPSMTDFQ IS........ ...... IL-1g_humanLILKKEDE.. .LGDRSIMFT VQNED..... ...... IL-1g_mouseLILKKKDE.. .NGDKSVMFT LTNLHQS... ...... IL-1b_humanVFLGGTK... .GGQDITDFT MQFVSS.... ...... IL-1b_mouseVFLGNNS... ..GQDIIDFT MESVSS.... ...... IL-1x_humanVSLTNMPD.. .EGVMVTKFY FQEDE..... ...... IL-1x_mouseVSLTNTPE.. .EPLIVTKFY FQEDQ..... ...... IL-1d_mouseVRLTQIPEDP AWDAPITDFY FQQCD..... ...... IL-1z_humanVGVTDKFE.. ..NRKHIEFS FQPVCKAEMS PSEVSD IL-1e_mouseLILTQELG.. ..EIFITDFE MIVVH..... ...... IL-1e_humanIILTSELG.. ..KSYNTAFE LNIND..... ...... IL-1a_human is SEQ ID NO: 5;IL-1a_mouse is SEQ ID NO: 6; IL-1g_human is SEQ ID NO: 7; IL-1g_mouse isSEQ ID NO: 8; IL-1b_human is SEQ ID NO: 9; IL-1b_mouse is SEQ ID NO: 10;IL-1x_human (IL-1RA) is SEQ ID NO: 11; IL-1x_mouse (IL-1RA) is SEQ IDNO: 12; IL-1d_mouse is SEQ ID NO: 13; IL-1e_mouse is SEQ ID NO: 14; andIL-1e_human is SEQ ID NO: 15.

TABLE 3 Relationship between various IL-1 family members, % identity forproteins: h1a m1a h1b m1b h1RA m1RA h1g m1g h1e m1e m1d h1z h1a m1a 54h1b 21 20 m1b 20 20 78 h1RA 18 16 25 26 m1RA 21 18 27 29 77 h1g 13 16 1516 18 17 m1g 15 17 15 15 15 15 63 h1e 17 16 21 18 30 29 14 12 m1e 18 1622 22 25 27 15 16 46 m1d 20 19 26 27 45 45 15 13 28 28 h1z 13 20 22 2426 28 11 13 22 18 20Comparison of the sequences will also provide an evolutionary tree. Thiscan be generated, e.g., using the TreeView program in combination withthe ClustalX analysis software program. See Thompson, et al. Nuc. AcidsRes. 25:4876-4882; and TreeView, Page, IBLS, University of Glasgow,e-mail rpage@bio.gla.ac.uk;http://taxonomy.zoology.gla.ac.uk.rod.treeview.html.

β conformation boundaries for IL-1ζ (SEQ ID NO: 2) are approximately: β1lys58-asp64; β2 val69-ser74; β3 asn76-val80; β4 phe91-ser96; β5ser107-val113; β6 phe118-lys126; β7 pro131-lys136; β8 phe154-val161; β9ser163-ser169; β10 phe176-ser180; β11 glu185-gln204; and β12phe201-gln204. The presence of amino acid residues between βconformations β4 and β5 are characteristic of IL-1 agonists. IL-1 familymolecules have highly conserved residues in the region encompassing βconformations β9 and β10. Segments beginning or ending at theseboundaries will be particularly interesting.

Various sites for interaction with receptor are: Site A includesresidues corresponding to positions of SEQ ID NO: 2 numbered 63-66,72-74, 78, 80-87, 181-186, and 202 and 204; Site B includes residuescorresponding to positions numbered 53-56, 58, 95-103, 159, 161-164,205, and 207; and Site C includes residues corresponding to positionsnumbered 127-153. See, e.g., U.S. Ser. No. 09/097,976, which isincorporated herein by reference.

As used herein, the term IL-1ζ shall be used to describe a proteincomprising a protein or peptide segment having or sharing an amino acidsequence shown in Table 1, or a substantial fragment thereof. Theinvention also includes protein variations of the IL-1ζ allele whosesequence is provided, e.g., a mutein agonist or antagonist. Typically,such agonists or antagonists will exhibit less than about 10% sequencedifferences, and thus will often have between 1- and 11-foldsubstitutions, e.g., 2-, 3-, 5-, 7-fold, and others. It also encompassesallelic and other variants, e.g., natural polymorphic variants, of theprotein described. “Natural” as used herein means unmodified byartifice. Typically, it will bind to its corresponding biologicalreceptor with high affinity, e.g., at least about 100 nM, usually betterthan about 30 nM, preferably better than about 10 nM, and morepreferably at better than about 3 nM. The term shall also be used hereinto refer to related naturally occurring forms, e.g., alleles,polymorphic variants, and metabolic variants of the mammalian protein.

This invention also encompasses proteins or peptides having substantialamino acid sequence homology with the amino acid sequences in Table 1.It will include sequence variants with relatively few substitutions,e.g., typically less than about 3-5.

A substantial polypeptide “fragment”, or “segment”, is a stretch ofamino acid residues of at least about 8 amino acids, generally at least10 amino acids, more generally at least 12 amino acids, often at least14 amino acids, more often at least 16 amino acids, typically at least18 amino acids, more typically at least 20 amino acids, usually at least22 amino acids, more usually at least 24 amino acids, preferably atleast 26 amino acids, more preferably at least 28 amino acids, and, inparticularly preferred embodiments, at least about 30 or more aminoacids, e.g., 35, 40, 45, 50, 60, 70, 80, etc. Sequences of segments ofdifferent proteins can be compared to one another over appropriatelength stretches.

Amino acid sequence homology, or sequence identity, is determined byoptimizing residue matches, if necessary, by introducing gaps asrequired. See, e.g., Needleham, et al., (1970) J. Mol. Biol. 48:443-453;Sankoff, et al. (1983) chapter one in Time Warps, String Edits, andMacromolecules: The Theory and Practice of Sequence Comparison,Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif.; and the University of WisconsinGenetics Computer Group (GCG), Madison, Wis.; each of which isincorporated herein by reference. This changes when consideringconservative substitutions as matches. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid;asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. Homologous amino acid sequences are intended toinclude natural allelic and interspecies variations in the cytokinesequence. Typical homologous proteins or peptides will have from 50-100%homology (if gaps can be introduced), to 60-100% homology (ifconservative substitutions are included) with an amino acid sequencesegments of Table 1. Homology measures will be at least about 70%,generally at least 76%, more generally at least 81%, often at least 85%,more often at least 88%, typically at least 90%, more typically at least92%, usually at least 94%, more usually at least 95%, preferably atleast 96%, and more preferably at least 97%, and in particularlypreferred embodiments, at least 98% or more. The degree of homology willvary with the length of the compared segments. Homologous proteins orpeptides, such as the allelic variants, will share most biologicalactivities with the embodiments described in Table 1. As used herein,the term “biological activity” is used to describe, without limitation,effects on inflammatory responses and/or innate immunity. For example,it may, like IL-1γ, exhibit synergistic induction by splenocytes ofIFN-γ in combination with IL-12 or IL-2, with or without anti-type I oranti-type II IL-1 receptor antibodies, or more structural properties asreceptor binding and cross-reactivity with antibodies raised against thesame or a polymorphic variant of a mammalian IL-1ζ.

The terms ligand, agonist, antagonist, and analog of, e.g., IL-1ζ,include molecules that modulate the characteristic cellular responses toIL-1ζ or IL-1ζ-like proteins, as well as molecules possessing the morestandard structural binding competition features of ligand-receptorinteractions, e.g., where the receptor is a natural receptor or anantibody. The cellular responses likely are mediated through binding ofIL-1ζ to cellular receptors related to, but possibly distinct from, thetype I or type II IL-1 receptors. Also, a ligand is a molecule whichserves either as a natural ligand to which said receptor, or an analogthereof, binds, or a molecule which is a functional analog of thenatural ligand. The functional analog may be a ligand with structuralmodifications, or may be a wholly unrelated molecule which has amolecular shape which interacts with the appropriate ligand bindingdeterminants. The ligands may serve as agonists or antagonists, see,e.g., Goodman, et al. (eds. 1990) Goodman & Gilman's: ThePharmacological Bases of Therapeutics, Pergamon Press, New York.

Rational drug design may also be based upon structural studies of themolecular shapes of a receptor or antibody and other effectors orligands. Effectors may be other proteins which mediate other functionsin response to ligand binding, or other proteins which normally interactwith the receptor. One means for determining which sites interact withspecific other proteins is a physical structure determination, e.g.,x-ray crystallography or 2 dimensional NMR techniques. These willprovide guidance as to which amino acid residues form molecular contactregions. For a detailed description of protein structural determination,see, e.g., Blundell and Johnson (1976) Protein Crystallography, AcademicPress, New York, which is hereby incorporated herein by reference.

II. Activities

The IL-1ζ polypeptides will have a number of different biologicalactivities, e.g., in the immune system, and will include inflammatoryfunctions or other innate immunity responses. The IL-1ζ polypeptides arehomologous to other IL-1 proteins, but each have structural differences.For example, a human IL-1γ gene coding sequence probably has about 70%identity with the nucleotide coding sequence of mouse IL-1γ, and similarmeasures of similarity will likely apply to the IL-1ζ. At the amino acidlevel, there is also likely to be about 60% identity. This level ofsimilarity suggests that the new IL-1ζ proteins are related to the otherIL-1α, IL-1β, IL-1RA, IL-1γ, IL-1δ, and IL-1ε.

The mouse IL-1γ molecule has the ability to stimulate IFN-γ productionwhich augments NK activity in spleen cells. See Okamura, et al. (1995)Nature 378:88-91.

The activities of the mouse IL-1α, IL-1β, and IL-1γ have been comparedas to their ability to induce IFN-γ, alone or in combination with IL-2or IL-12 in SCID splenocytes and purified NK cells. See Hunter, et al.(1995) J. Immunol. 155:4347-4354; and Bancroft, et al. (1991) Immunol.Revs. 124:5-24. The IL-1γ was found to be much more potent instimulating IFN-1γ than either IL-1α or IL-1β. IL-1ζ and agonists orantagonists should have related activities to these or to the other newIL-1δ and IL-1ε, typically affecting similar immune functions, includinginflammatory responses.

In IL-2 activated NK cells, IFN-γ production is blocked by the additionof anti-IL-1β antibodies. See Hunter, et al. (1995). However, mouseIL-1γ can overcome this block and induce IFN-γ. This is the onlycytokine known to be able to do this. In addition, in vivo,administration of mouse IL-1γ to mice infected with the parasite T.Cruzi significantly decreases parasitemia.

The present disclosure also describes new assays for activitiespredicted for the IL-1ζ molecules. Corresponding activities should befound in other mammalian systems, including primates or rodents. It islikely that the new primate IL-1-like molecules produced by similarrecombinant means to the human IL-1γ protein should exhibit a biologicalactivity of modulating lymphocyte cells in production of IFN-γ. Seeassays described, e.g., in de Waal Malefyt, et al., in de Vries and deWaal Malefyt (eds. 1995) “Interleukin-10” Landes Co., Austin, Tex.Furthermore, there is substantial likelihood of synergy with other IL-1or IL-12 related agonists or antagonists. It is likely that thereceptors, which are expected to include multiple different polypeptidechains, exhibit species specificity for their corresponding ligands. TheIL-1α and IL-1β ligands both signal through heterodimeric receptors.

III. Nucleic Acids

This invention contemplates use of isolated nucleic acid or fragments,e.g., which encode this or a closely related protein, or fragmentsthereof, e.g., to encode a biologically active correspondingpolypeptide. The term “isolated nucleic acid or fragments” as usedherein means a nucleic acid, e.g., a DNA or RNA molecule, that is notimmediately contiguous when present in the naturally occurring genome ofthe organism from which it is derived. Thus, the term describes, e.g., anucleic acid that is incorporated into a vector, such as a plasmid orviral vector; a nucleic acid that is incorporated into the genome of aheterologous cell (or the genome of homologous cell, but at a sitedifferent from that at which it normally occurs); and a nucleic acidthat exists as a separate molecule, e.g., a DNA fragment produced by PCRamplification or restriction enzyme digestion, or an RNA moleculeproduced by in vitro transcription. The term also describes arecombinant (i.e., genetically engineered) nucleic acid that forms partof a hybrid gene encoding additional polypeptide sequences that can beused, e.g., in the production of a fusion protein. In addition, thisinvention embodies virtually any engineered or nucleic acid moleculecreated by artifice that encodes a biologically active protein orpolypeptide having characteristic IL-1ζ activity.

Typically, the nucleic acid is capable of hybridizing, under appropriateconditions, with a nucleic acid sequence segment shown in Table 1. Saidbiologically active protein or polypeptide can be a full length protein,or fragment, and will typically have a segment of amino acid sequencehighly homologous to one shown in Table 1. Further, this inventioncovers the use of isolated or recombinant nucleic acid, or fragmentsthereof, which encode proteins having fragments which are homologous tothe newly disclosed IL-1-like proteins. The isolated nucleic acids canhave the respective regulatory sequences in the 5′ and 3′ flanks, e.g.,promoters, enhancers, poly-A addition signals, and others from thenatural gene.

An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA, or amixed polymer, which is substantially pure, e.g., separated from othercomponents which naturally accompany a native sequence, such asribosomes, polymerases, and flanking genomic sequences from theoriginating species. The term embraces a nucleic acid sequence which hasbeen removed from its naturally occurring environment, and includesrecombinant or cloned DNA isolates, which are thereby distinguishablefrom naturally occurring compositions, and chemically synthesizedanalogs or analogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule,either completely or substantially pure.

An isolated nucleic acid will generally be a homogeneous composition ofmolecules, but will, in some embodiments, contain heterogeneity,preferably minor. This heterogeneity is typically found at the polymerends or portions not critical to a desired biological function oractivity.

A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence. Typically this intervention involves in vitromanipulation, although under certain circumstances it may involve moreclassical animal breeding techniques. Alternatively, it can be a nucleicacid made by generating a sequence comprising fusion of two fragmentswhich are not naturally contiguous to each other, but is meant toexclude products of nature, e.g., naturally occurring mutants as foundin their natural state. Thus, e.g., products made by transforming cellswith an unnaturally occurring vector is encompassed, as are nucleicacids comprising sequence derived using a synthetic oligonucleotideprocess. Such a process is often done to replace a codon with aredundant codon encoding the same or a conservative amino acid, whiletypically introducing or removing a restriction enzyme sequencerecognition site. Alternatively, the process is performed to jointogether nucleic acid segments of desired functions to generate a singlegenetic entity comprising a desired combination of functions not foundin the commonly available natural forms, e.g., encoding a fusionprotein. Restriction enzyme recognition sites are often the target ofsuch artificial manipulations, but other site specific targets, e.g.,promoters, DNA replication sites, regulation sequences, controlsequences, or other useful features may be incorporated by design. Asimilar concept is intended for a recombinant, e.g., fusion,polypeptide. This will include a dimeric repeat. Specifically includedare synthetic nucleic acids which, by genetic code redundancy, encodesimilar polypeptides to fragments of the IL-1ζ and fusions of sequencesfrom various different interleukin or related molecules, e.g., growthfactors.

A “fragment” in a nucleic acid context is a contiguous segment of atleast about 17 nucleotides, generally at least 21 nucleotides, moregenerally at least 25 nucleotides, ordinarily at least 30 nucleotides,more ordinarily at least 35 nucleotides, often at least 39 nucleotides,more often at least 45 nucleotides, typically at least 50 nucleotides,more typically at least 55 nucleotides, usually at least 60 nucleotides,more usually at least 66 nucleotides, preferably at least 72nucleotides, more preferably at least 79 nucleotides, and inparticularly preferred embodiments will be at least 85 or morenucleotides including, e.g., 100, 150, 200, 250, etc. Preferredembodiments will exhibit a plurality of distinct, e.g., nonoverlapping,segments of the specified length. Typically, the plurality will be atleast two, more usually at least three, and preferably 5, 7, or evenmore. While the length minima are provided, longer lengths, of varioussizes, may be appropriate, e.g., one of length 7, and two of length 12.Typically, fragments of different genetic sequences can be compared toone another over appropriate length stretches, particularly definedsegments such as the domains described below.

A nucleic acid which codes for an IL-1ζ will be particularly useful toidentify genes, mRNA, and cDNA species which code for itself or closelyrelated proteins, as well as DNAs which code for polymorphic, allelic,or other genetic variants, e.g., from different individuals or relatedspecies. Preferred probes for such screens are those regions of theinterleukin which are conserved between different polymorphic variantsor which contain nucleotides which lack specificity, and will preferablybe full length or nearly so. In other situations, polymorphic variantspecific sequences will be more useful.

This invention further covers recombinant nucleic acid molecules andfragments having a nucleic acid sequence identical to or highlyhomologous to the isolated DNA set forth herein. In particular, thesequences will often be operably linked to DNA segments which controltranscription, translation, and DNA replication. These additionalsegments typically assist in expression of the desired nucleic acidsegment.

Homologous nucleic acid sequences, when compared to one another or Table1 sequences, exhibit significant similarity. The standards for homologyin nucleic acids are either measures for homology generally used in theart by sequence comparison or based upon hybridization conditions.Comparative hybridization conditions are described in greater detailbelow.

Substantial identity in the nucleic acid sequence comparison contextmeans either that the segments, or their complementary strands, whencompared, are identical when optimally aligned, with appropriatenucleotide insertions or deletions, in at least about 60% of thenucleotides, generally at least 66%, ordinarily at least 71%, often atleast 76%, more often at least 80%, usually at least 84%, more usuallyat least 88%, typically at least 91%, more typically at least about 93%,preferably at least about 95%, more preferably at least about 96 to 98%or more, and in particular embodiments, as high at about 99% or more ofthe nucleotides, including, e.g., segments encoding structural domainssuch as the segments described below. Alternatively, substantialidentity will exist when the segments will hybridize under selectivehybridization conditions, to a strand or its complement, typically usinga sequence derived from Table 1. Typically, selective hybridization willoccur when there is at least about 55% homology over a stretch of atleast about 14 nucleotides, more typically at least about 65%,preferably at least about 75%, and more preferably at least about 90%.See, Kanehisa (1984) Nuc. Acids Res. 12:203-213. The length of homologycomparison, as described, may be over longer stretches, and in certainembodiments will be over a stretch of at least about 17 nucleotides,generally at least about 20 nucleotides, ordinarily at least about 24nucleotides, usually at least about 28 nucleotides, typically at leastabout 32 nucleotides, more typically at least about 40 nucleotides,preferably at least about 50 nucleotides, and more preferably at leastabout 75 to 100 or more nucleotides.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optical alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith and Waterman (1981) Adv. Appl.Math. 2:482, by the homology alignment algorithm of Needlman and Wunsch(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally Ausubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendrogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described byHiggins and Sharp (1989) CABIOS 5:151-153. The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described Altschul, et al. (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http:www.ncbi.nlm.nih.gov/). Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul, et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extension of the word hitsin each direction are halted when: the cumulative alignment score fallsoff by the quantity X from its maximum achieved value; the cumulativescore goes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Nat'l Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat'l Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences of polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

Stringent conditions, in referring to homology in the hybridizationcontext, will be stringent combined conditions of salt, temperature,organic solvents, and other parameters typically controlled inhybridization reactions. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30° C., more usually in excessof about 37° C., typically in excess of about 45° C., more typically inexcess of about 55° C., preferably in excess of about 65° C., and morepreferably in excess of about 70° C. Stringent salt conditions willordinarily be less than about 500 mM, usually less than about 400 mM,more usually less than about 300 mM, typically less than about 200 mM,preferably less than about 100 mM, and more preferably less than about80 mM, even down to less than about 20 mM. However, the combination ofparameters is much more important than the measure of any singleparameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol.31:349-370, which is hereby incorporated herein by reference.Hybridization under stringent conditions should give a background of atleast 2-fold over background, preferably at least 3-5 or more.

The isolated DNA can be readily modified by nucleotide substitutions,nucleotide deletions, nucleotide insertions, and inversions ofnucleotide stretches. These modifications result in novel DNA sequenceswhich encode this protein or its derivatives. These modified sequencescan be used to produce mutant proteins (muteins) or to enhance theexpression of variant species. Enhanced expression may involve geneamplification, increased transcription, increased translation, and othermechanisms. Such mutant IL-1-like derivatives include predetermined orsite-specific mutations of the protein or its fragments, includingsilent mutations using genetic code degeneracy. “Mutant IL-1ζ” as usedherein encompasses a polypeptide otherwise falling within the homologydefinition of the IL-1ζ as set forth above, but having an amino acidsequence which differs from that of other IL-1-like proteins as found innature, whether by way of deletion, substitution, or insertion. Inparticular, “site specific mutant IL-1ζ” encompasses a protein havingsubstantial homology with a protein of Table 1, and typically sharesmost of the biological activities of the form disclosed herein.

Although site specific mutation sites are predetermined, mutants neednot be site specific. Mammalian IL-1ζ mutagenesis can be achieved bymaking amino acid insertions or deletions in the gene, coupled withexpression. Substitutions, deletions, insertions, or combinations may begenerated to arrive at a final construct. Insertions include amino- orcarboxy-terminal fusions. Random mutagenesis can be conducted at atarget codon and the expressed mammalian IL-1ζ mutants can then bescreened for the desired activity. Methods for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown in the art, e.g., by M13 primer mutagenesis. See also Sambrook, etal. (1989) and Ausubel, et al. (1987 and periodic Supplements).

The mutations in the DNA normally should not place coding sequences outof reading frames and preferably will not create complementary regionsthat could hybridize to produce secondary mRNA structure such as loopsor hairpins.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence.

Polymerase chain reaction (PCR) techniques can often be applied inmutagenesis. Alternatively, mutagenesis primers are commonly usedmethods for generating defined mutations at predetermined sites. See,e.g., Innis, et al. (eds. 1990) PCR Protocols: A Guide to Methods andApplications Academic Press, San Diego, Calif.; and Dieffenbach andDveksler (1995; eds.) PCR Primer: A Laboratory Manual Cold Spring HarborPress, CSH, NY.

IV. Proteins, Peptides

As described above, the present invention encompasses mammalian IL-1ζ,e.g., whose sequences are disclosed in Table 1, and described above.Allelic and other variants are also contemplated, including, e.g.,fusion proteins combining portions of such sequences with others,including epitope tags and functional domains.

The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these rodent proteins.A heterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. Thus, the fusionproduct of a growth factor with an interleukin is a continuous proteinmolecule having sequences fused in a typical peptide linkage, typicallymade as a single translation product and exhibiting properties derivedfrom each source peptide. A similar concept applies to heterologousnucleic acid sequences.

In addition, new constructs may be made from combining similarfunctional or structural domains from other related proteins, e.g.,growth factors or other cytokines. For example, receptor-binding orother segments may be “swapped” between different new fusionpolypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992,each of which is incorporated herein by reference. Thus, new chimericpolypeptides exhibiting new combinations of specificities will resultfrom the functional linkage of receptor-binding specificities. Forexample, the receptor binding domains from other related ligandmolecules may be added or substituted for other domains of this orrelated proteins. The resulting protein will often have hybrid functionand properties. For example, a fusion protein may include a targetingdomain which may serve to provide sequestering of the fusion protein toa particular organ, e.g., a ligand portions which is specifically boundby spleen cells and would serve to accumulate in the spleen.

Candidate fusion partners and sequences can be selected from varioussequence data bases, e.g., GenBank, c/o NCBI; and BCG, University ofWisconsin Biotechnology Computing Group, Madison, Wis., which are eachincorporated herein by reference.

The present invention particularly provides muteins which act asagonists or antagonists of the IL-1ζ. Structural alignment of primateIL-1ζ and other members of the IL-1 family show conservedfeatures/residues, particularly 12 β strands folded into a β-trefoilfold. The 12 IL-1ζ β strand domains are recited, respectively, about: β1lys58-asp64; β2 val69-ser74; β3 asn76-val80; β4 phe91-ser96; β5ser107-val113; β6 phe118-lys126; β7 pro131-lys136; β8 phe154-val161; β9ser163-ser169; β10 phe176-ser180; β11 glu185-gln204; and β12phe201-gln204. The presence of amino acid residues between βconformations β4 and β5 are characteristic of IL-1 agonists. IL-1 familymolecules have highly conserved residues in the region encompassing βconformations β9 and β10.

Alignment of the primate IL-1ζ with other members of the IL-1 familyindicates that the β conformations correspond to similar sequences inother IL-1 family members. See also, Bazan, et al. (1996) Nature379:591; Lodi, et al. (1994) Science 263:1762-1766; Sayle andMilner-White (1995) TIBS 20:374-376; and Gronenberg, et al. (1991)Protein Engineering 4:263-269.

The IL-1α and IL-1β ligands bind an IL-1 receptor type I as the primaryreceptor and this complex then forms a high affinity receptor complexwith the IL-1 receptor type III. Such receptor subunits are probablyshared with the new IL-1 family members.

The mouse IL-1γ does not bind to the known mouse IL-1 receptor types I,II (decoy receptor), or III. In addition, the mouse IGIF biologicalactivity cannot be blocked with anti-type I, II, or III antibodies. Thissuggests that the related mouse IGIF binds to receptors related to theIL-1 receptors already isolated, but not yet identified as receptors forthe IGIF.

The solved structures for IL-1β, the natural IL-1 receptor antagonist(IL-1Ra), and a co-structure of IL-1Ra/IL-1 receptor type I, however,suggest how to make a primate antagonist for IL-1ζ (see, e.g., accessionnumbers: U65590, gbU19844, gbU19845, gi2173679, gi2170133, gi2172939,gbM15300, gbM28983, gbU65590, gbM74294, embX04964, gi2169698, gi2169368emb270047, gi914939, gi220782, embX52731, embX56972 and embX12497, forvarious species examples of IL-1 family members). Structural analyses ofthe mature primate IL-1ζ suggest that its β-trefoil structures contactthe IL-1 receptor over three binding sites (designated A, B and C).Sites A and C bind to the first receptor subunit (alpha) of IL-1 whilesite B binds the IL-1 second receptor subunit (beta). Homology sequencecomparison of the IL-1 family members reveals that the only knownantagonist to IL-1 receptor (IL-1x, or IL-1Ra; Table 2) is missing anamino acid domain bounded by the β4 and β5 strands. This domain maps toa portion of site B in primate IL-1ζ (Table 2) that binds to the IL-1second receptor subunit, suggesting that its absence confers antagonistactivity as evidenced by homology comparison among other IL-1 familymembers. This loop portion of contact site B spans approximately 7-10amino residues, while in IL-1RA the loop is “cut off” with only 2residues remaining. Therefore, IL-1RA binds normally to receptor type I,but cannot interact with receptor type III. This makes IL-1RA into aneffective IL-1 antagonist.

The corresponding location in primate IL-1ζ (between β4 and β5) definesa domain that forms a polypeptide loop which is part of a primarybinding segment to the IL-1 receptor type. The loop is defined, forIL-1ζ, approximately by amino residues ser100-gly106 of SEQ ID NO: 2.Accordingly, IL-1ζ antagonist activity should be generated by removalall or an appropriate portion of a corresponding portion of amino acidslocated between β4 and β5. This suggests that analogous modifications tothe loop between the β4 and the β5 strands will lead to variants withpredictable biological activities. With mouse IL-1RA, it was shown thatreplacement of the mouse IL-1RA residues with those mouse IL-1β residuesintroduced IL-1 activity to the IL-1RA variant (IL-1RA could then bindtype III receptor). Similar substitutions should establish that type IIIreceptor can probably be used by primate IL-1ζ or muteins.

Sites A and C should mediate binding of IL-1ζ to the first IL-1 receptorsubunit, e.g., an alpha receptor subunit. Site A contacts correspond inIL-1ζ to amino residues corresponding to positions of SEQ ID NO: 2numbered about 63-66, 72-74, 78, 80-87, 181-186, and 202 and 204; Site Bincludes residues corresponding to positions numbered about 53-56, 58,95-103, 159, 161-164, 205, and 207; and Site C includes residuescorresponding to positions numbered about 127-153. See, e.g., U.S. Ser.No. 09/097,976, which is incorporated herein by reference.

Similar variations in other species counterparts of IL-1ζ ligandsequence, e.g., in the corresponding regions, should provide similarinteractions with receptor. Substitutions with either mouse sequences orhuman sequences are indicated. Conversely, conservative substitutionsaway from the receptor binding interaction regions will probablypreserve most biological activities.

“Derivatives” of the mammalian IL-1ζ include amino acid sequencemutants, glycosylation variants, metabolic derivatives and covalent oraggregative conjugates with other chemical moieties. Covalentderivatives can be prepared by linkage of functionality's to groupswhich are found in the IL-1ζ amino acid side chains or at the N- orC-termini, e.g., by means which are well known in the art. Thesederivatives can include, without limitation, aliphatic esters or amidesof the carboxyl terminus, or of residues containing carboxyl sidechains, O-acyl derivatives of hydroxyl group-containing residues, andN-acyl derivatives of the amino terminal amino acid or amino-groupcontaining residues, e.g., lysine or arginine. Acyl groups are selectedfrom the group of alkyl-moieties including C3 to C18 normal alkyl,thereby forming alkanoyl aroyl species.

In particular, glycosylation alterations are included, e.g., made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. Particularlypreferred means for accomplishing this are by exposing the polypeptideto glycosylating enzymes derived from cells which normally provide suchprocessing, e.g., mammalian glycosylation enzymes. Deglycosylationenzymes are also contemplated. Also embraced are versions of the sameprimary amino acid sequence which have other minor modifications,including phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine.

A major group of derivatives are covalent conjugates of the interleukinor fragments thereof with other proteins of polypeptides. Thesederivatives can be synthesized in recombinant culture such as N- orC-terminal fusions or by the use of agents known in the art for theirusefulness in cross-linking proteins through reactive side groups.Preferred derivatization sites with cross-linking agents are at freeamino groups, carbohydrate moieties, and cysteine residues.

Fusion polypeptides between the interleukin and other homologous orheterologous proteins are also provided. Homologous polypeptides may befusions between different growth factors, resulting in, for instance, ahybrid protein exhibiting ligand specificity for multiple differentreceptors, or a ligand which may have broadened or weakened specificityof binding to its receptor. Likewise, heterologous fusions may beconstructed which would exhibit a combination of properties oractivities of the derivative proteins. Typical examples are fusions of areporter polypeptide, e.g., luciferase, with a segment or domain of areceptor, e.g., a ligand-binding segment, so that the presence orlocation of a desired ligand may be easily determined. See, e.g., Dull,et al., U.S. Pat. No. 4,859,609, which is hereby incorporated herein byreference. Other gene fusion partners include glutathione-S-transferase(GST), bacterial β-galactosidase, trpE, Protein A, β-lactamase, alphaamylase, alcohol dehydrogenase, and yeast alpha mating factor. See,e.g., Godowski, et al. (1988) Science 241:812-816.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence.

Such polypeptides may also have amino acid residues which have beenchemically modified by phosphorylation, sulfonation, biotinylation, orthe addition or removal of other moieties, particularly those which havemolecular shapes similar to phosphate groups. In some embodiments, themodifications will be useful labeling reagents, or serve as purificationtargets, e.g., affinity ligands.

Fusion proteins will typically be made by either recombinant nucleicacid methods or by synthetic polypeptide methods. Techniques for nucleicacid manipulation and expression are described generally, e.g., inSambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.),Vols. 1-3, Cold Spring Harbor Laboratory, and Ausubel, et al. (eds. 1987and periodic supplements) Current Protocols in Molecular Biology,Greene/Wiley, New York, which are each incorporated herein by reference.Techniques for synthesis of polypeptides are described, e.g., inMerrifield (1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986)Science 232: 341-347; and Atherton, et al. (1989) Solid Phase PeptideSynthesis: A Practical Approach, IRL Press, Oxford; each of which isincorporated herein by reference. See also, Dawson, et al. (1994)Science 266:776-779 for methods to make larger polypeptides.

In another embodiment, the present invention relates to substantiallypurified peptide fragments of IL-1ζ that block binding between IL-1family members and a target receptor. Such peptide fragments couldrepresent research and diagnostic tools in the study of inflammatoryreactions to antigenic challenge and the development of more effectiveanti-inflammatory therapeutics. In addition, pharmaceutical compositionscomprising isolated and purified peptide fragments of IL-1ζ mayrepresent effective anti-inflammatory therapeutics.

The term “substantially purified” as used herein refers to a molecule,such as a peptide that is substantially free of other proteins, lipids,carbohydrates, nucleic acids, or other biological materials with whichit is naturally associated. For example, a substantially pure molecule,such as a polypeptide, can be at least 60%, by dry weight, the moleculeof interest. One skilled in the art can purify IL-1ζ peptides usingstandard protein purification methods and the purity of the polypeptidescan be determined using standard methods including, e.g., polyacrylamidegel electrophoresis (e.g., SDS-PAGE), column chromatography (e.g., highperformance liquid chromatography (HPLC)), and amino-terminal amino acidsequence analysis.

The invention relates not only to fragments of naturally-occurringIL-1ζ, but also to IL-1ζ mutants and chemically synthesized derivativesof IL-1ζ that block binding between IL-1 family members and a targetreceptor.

For example, changes in the amino acid sequence of IL-1ζ arecontemplated in the present invention. IL-1ζ can be altered by changingthe nucleic acid sequence encoding the protein. Preferably, onlyconservative amino acid alterations are undertaken, using amino acidsthat have the same or similar properties. Illustrative amino acidsubstitutions include the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine,glutamine, or glutamate; methionine to leucine or isoleucine;phenylalanine to tyrosine, leucine or methionine; serine to threonine;threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan orphenylalanine; valine to isoleucine or leucine.

Additionally, other variants and fragments of IL-1ζ can be used in thepresent invention. Variants include analogs, homologues, derivatives,muteins, and mimetics of IL-1ζ that retain the ability to block bindingbetween IL-1 family members and a target receptor. Fragments of theIL-1ζ refer to portions of the amino acid sequence of IL-1ζ as definedin SEQ ID NO: 2 that also retain this ability. The variants andfragments can be generated directly from IL-1ζ itself by chemicalmodification, by proteolytic enzyme digestion, or by combinationsthereof. Additionally, genetic engineering techniques, as well asmethods of synthesizing polypeptides directly from amino acid residues,can be employed.

Non-peptide compounds that mimic the binding and function of IL-1ζ(“mimetics”) can be produced by the approach outlined in Saragovi, etal. (1991) Science 253:792-795. Mimetics are molecules which mimicelements of protein secondary structure. See, e.g., Johnson et al.,“Peptide Turn Mimetics,” in Pezzuto, et al. (eds. 1993) Biotechnologyand Pharmacy, Chapman and Hall, New York. The underlying rationalebehind the use of peptide mimetics is that the peptide backbone ofproteins exists chiefly to orient amino acid side chains in such a wayas to facilitate molecular interactions. For the purposes of the presentinvention, appropriate mimetics can be considered to be the equivalentof IL-1ζ.

Variants and fragments also can be created by recombinant techniquesemploying genomic or cDNA cloning methods. Site-specific andregion-directed mutagenesis techniques can be employed. See, e.g., vol.1, ch. 8 in Ausubel, et al. (eds. 1989 and periodic updates) CurrentProtocols in Molecular Biology Wiley and Sons; and Oxender and Fox(eds.) Protein Engineering Liss, Inc. In addition, linker-scanning andPCR-mediated techniques can be employed for mutagenesis. See, e.g.,Erlich (ed. 1989) PCR Technology Stockton Press. Protein sequencing,structure and modeling approaches for use with the above techniques aredisclosed, e.g., in Oxender and Fox (eds.) Protein Engineering Liss,Inc; and Ausubel, et al. (eds. 1989 and periodic updates) CurrentProtocols in Molecular Biology Wiley and Sons.

This invention also contemplates the use of derivatives of IL-1ζ otherthan variations in amino acid sequence or glycosylation. Suchderivatives may involve covalent or aggregative association withchemical moieties. These derivatives generally fall into three classes:(1) salts, (2) side chain and terminal residue covalent modifications,and (3) adsorption complexes, e.g., with cell membranes. Such covalentor aggregative derivatives are useful as immunogens, as reagents inimmunoassays, or in purification methods such as for affinitypurification of a receptor or other binding molecule, e.g., an antibody.For example, an IL-1ζ ligand can be immobilized by covalent bonding to asolid support such as cyanogen bromide-activated SEPHAROSE, by methodswhich are well known in the art, or adsorbed onto polyolefin surfaces,with or without glutaraldehyde cross-linking, for use in the assay orpurification of IL-1ζ receptor, antibodies, or other similar molecules.The IL-1ζ can also be labeled with a detectable group, e.g.,radio-iodinated by the chloramine T procedure, covalently bound to rareearth chelates, or conjugated to another fluorescent moiety for use indiagnostic assays.

An IL-1ζ of this invention can be used as an immunogen for theproduction of antisera or antibodies specific, e.g., capable ofdistinguishing between other IL-1 family members and an IL-1ζ, for theinterleukin or fragments thereof. The purified interleukin can be usedto screen monoclonal antibodies or antigen-binding fragments prepared byimmunization with various forms of impure preparations containing theprotein. In particular, the term “antibodies” also encompasses antigenbinding fragments of natural antibodies. The purified interleukin canalso be used as a reagent to detect antibodies generated in response tothe presence of elevated levels of expression, or immunologicaldisorders which lead to antibody production to the endogenous cytokine.Additionally, IL-1ζ fragments may also serve as immunogens to producethe antibodies of the present invention, as described immediately below.For example, this invention contemplates antibodies having bindingaffinity to or being raised against the amino acid sequence shown inTable 1, fragments thereof, or homologous peptides. In particular, thisinvention contemplates antibodies having binding affinity to, or havingbeen raised against, specific fragments which are predicted to be, oractually are, exposed at the exterior protein surface of the nativecytokine.

The blocking of physiological response to these interleukins may resultfrom the inhibition of binding of the ligand to the receptor, likelythrough competitive inhibition. Thus, in vitro assays of the presentinvention will often use antibodies or ligand binding segments of theseantibodies, or fragments attached to solid phase substrates. Theseassays will also allow for the diagnostic determination of the effectsof either binding region mutations and modifications, or ligandmutations and modifications, e.g., ligand analogs.

This invention also contemplates the use of competitive drug screeningassays, e.g., where neutralizing antibodies to the interleukin orfragments compete with a test compound for binding to a receptor orantibody. In this manner, the neutralizing antibodies or fragments canbe used to detect the presence of a polypeptide which shares one or morebinding sites to a receptor and can also be used to occupy binding siteson a receptor that might otherwise bind an interleukin.

V. Making Nucleic Acids and Protein

DNA which encodes the protein or fragments thereof can be obtained bychemical synthesis, screening cDNA libraries, or by screening genomiclibraries prepared from a wide variety of cell lines or tissue samples.Natural sequences can be isolated using standard methods and thesequences provided herein, e.g., in Table 1. Other species counterpartscan be identified by hybridization techniques, or by various PCRtechniques, combined with or by searching in sequence databases.

This DNA can be expressed in a wide variety of host cells for thesynthesis of a full-length interleukin or fragments which can in turn,e.g., be used to generate polyclonal or monoclonal antibodies; forbinding studies; for construction and expression of modifiedagonist/antagonist molecules; and for structure/function studies. Eachvariant or its fragments can be expressed in host cells that aretransformed or transfected with appropriate expression vectors. Thesemolecules can be substantially free of protein or cellular contaminants,other than those derived from the recombinant host, and therefore areparticularly useful in pharmaceutical compositions when combined with apharmaceutically acceptable carrier and/or diluent. The protein, orportions thereof, may be expressed as fusions with other proteins.

Expression vectors are typically self-replicating DNA or RNA constructscontaining the desired receptor gene or its fragments, usually operablylinked to suitable genetic control elements that are recognized in asuitable host cell. These control elements are capable of effectingexpression within a suitable host. The specific type of control elementsnecessary to effect expression will depend upon the eventual host cellused. Generally, the genetic control elements can include a prokaryoticpromoter system or a eukaryotic promoter expression control system, andtypically include a transcriptional promoter, an optional operator tocontrol the onset of transcription, transcription enhancers to elevatethe level of mRNA expression, a sequence that encodes a suitableribosome binding site, and sequences that terminate transcription andtranslation. Expression vectors also usually contain an origin ofreplication that allows the vector to replicate independently of thehost cell.

The vectors of this invention include those which contain DNA whichencodes a protein, as described, or a fragment thereof encoding abiologically active equivalent polypeptide. The DNA can be under thecontrol of a viral promoter and can encode a selection marker. Thisinvention further contemplates use of such expression vectors which arecapable of expressing eukaryotic cDNA coding for such a protein in aprokaryotic or eukaryotic host, where the vector is compatible with thehost and where the eukaryotic cDNA coding for the receptor is insertedinto the vector such that growth of the host containing the vectorexpresses the cDNA in question. Usually, expression vectors are designedfor stable replication in their host cells or for amplification togreatly increase the total number of copies of the desirable gene percell. It is not always necessary to require that an expression vectorreplicate in a host cell, e.g., it is possible to effect transientexpression of the interleukin protein or its fragments in various hostsusing vectors that do not contain a replication origin that isrecognized by the host cell. It is also possible to use vectors thatcause integration of the protein encoding portion or its fragments intothe host DNA by recombination.

Vectors, as used herein, comprise plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles which enable theintegration of DNA fragments into the genome of the host. Expressionvectors are specialized vectors which contain genetic control elementsthat effect expression of operably linked genes. Plasmids are the mostcommonly used form of vector but all other forms of vectors which servean equivalent function and which are, or become, known in the art aresuitable for use herein. See, e.g., Pouwels, et al. (1985 andSupplements) Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., andRodriquez, et al. (eds.) Vectors: A Survey of Molecular Cloning Vectorsand Their Uses, Buttersworth, Boston, 1988, which are incorporatedherein by reference.

Transformed cells are cells, preferably mammalian, that have beentransformed or transfected with receptor vectors constructed usingrecombinant DNA techniques. Transformed host cells usually express thedesired protein or its fragments, but for purposes of cloning,amplifying, and manipulating its DNA, do not need to express the subjectprotein. This invention further contemplates culturing transformed cellsin a nutrient medium, thus permitting the interleukin to accumulate inthe culture. The protein can be recovered, either from the culture orfrom the culture medium.

For purposes of this invention, nucleic sequences are operably linkedwhen they are functionally related to each other. For example, DNA for apre-sequence or secretory leader is operably linked to a polypeptide ifit is expressed as a pre-protein or participates in directing thepolypeptide to the cell membrane or in secretion of the polypeptide. Apromoter is operably linked to a coding sequence if it controls thetranscription of the polypeptide; a ribosome binding site is operablylinked to a coding sequence if it is positioned to permit translation.Usually, operably linked means contiguous and in reading frame, however,certain genetic elements such as repressor genes are not contiguouslylinked but still bind to operator sequences that in turn controlexpression.

Suitable host cells include prokaryotes, lower eukaryotes, and highereukaryotes. Prokaryotes include both gram negative and gram positiveorganisms, e.g., E. coli and B. subtilis. Lower eukaryotes includeyeasts, e.g., S. cerevisiae and Pichia, and species of the genusDictyostelium. Higher eukaryotes include established tissue culture celllines from animal cells, both of non-mammalian origin, e.g., insectcells, and birds, and of mammalian origin, e.g., human, primates, androdents.

Prokaryotic host-vector systems include a wide variety of vectors formany different species. As used herein, E. coli and its vectors will beused generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 ormany of its derivatives. Vectors that can be used to express thereceptor or its fragments include, but are not limited to, such vectorsas those containing the lac promoter (pUC-series); trp promoter(pBR322-trp); Ipp promoter (the pIN-series); lambda-pP or pR promoters(pOTS); or hybrid promoters such as ptac (pDR540). See Brosius, et al.(1988) “Expression Vectors Employing Lambda-, trp-, lac-, andIpp-derived Promoters”, in Vectors: A Survey of Molecular CloningVectors and Their Uses, (eds. Rodriguez and Denhardt), Buttersworth,Boston, Chapter 10, pp. 205-236, which is incorporated herein byreference.

Lower eukaryotes, e.g., yeasts and Dictyostelium, may be transformedwith IL-1ζ sequence containing vectors. For purposes of this invention,the most common lower eukaryotic host is the baker's yeast,Saccharomyces cerevisiae. It will be used to generically represent lowereukaryotes although a number of other strains and species are alsoavailable. Yeast vectors typically consist of a replication origin(unless of the integrating type), a selection gene, a promoter, DNAencoding the receptor or its fragments, and sequences for translationtermination, polyadenylation, and transcription termination. Suitableexpression vectors for yeast include such constitutive promoters as3-phosphoglycerate kinase and various other glycolytic enzyme genepromoters or such inducible promoters as the alcohol dehydrogenase 2promoter or metallothionine promoter. Suitable vectors includederivatives of the following types: self-replicating low copy number(such as the YRp-series), self-replicating high copy number (such as theYEp-series); integrating types (such as the YIp-series), ormini-chromosomes (such as the YCp-series).

Higher eukaryotic tissue culture cells are normally the preferred hostcells for expression of the functionally active interleukin protein. Inprinciple, virtually any higher eukaryotic tissue culture cell line isworkable, e.g., insect baculovirus expression systems, whether from aninvertebrate or vertebrate source. However, mammalian cells arepreferred. Transformation or transfection and propagation of such cellshas become a routine procedure. Examples of useful cell lines includeHeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney(BRK) cell lines, insect cell lines, bird cell lines, and monkey (COS)cell lines. Expression vectors for such cell lines usually include anorigin of replication, a promoter, a translation initiation site, RNAsplice sites (if genomic DNA is used), a polyadenylation site, and atranscription termination site. These vectors also usually contain aselection gene or amplification gene. Suitable expression vectors may beplasmids, viruses, or retroviruses carrying promoters derived, e.g.,from such sources as from adenovirus, SV40, parvoviruses, vacciniavirus, or cytomegalovirus. Representative examples of suitableexpression vectors include pcDNA1; pCD, see Okayama, et al. (1985) Mol.Cell. Biol. 5:1136-1142; pMC1neo PolyA, see Thomas, et al. (1987) Cell51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.

For secreted proteins, an open reading frame usually encodes apolypeptide that consists of a mature or secreted product covalentlylinked at its N-terminus to a signal peptide. The signal peptide iscleaved prior to secretion of the mature, or active, polypeptide. Thecleavage site can be predicted with a high degree of accuracy fromempirical rules, e.g., von-Heijne (1986) Nucleic Acids Research14:4683-4690, and the precise amino acid composition of the signalpeptide does not appear to be critical to its function, e.g., Randall,et al. (1989) Science 243:1156-1159; Kaiser et al. (1987) Science235:312-317.

It will often be desired to express these polypeptides in a system whichprovides a specific or defined glycosylation pattern. In this case, theusual pattern will be that provided naturally by the expression system.However, the pattern will be modifiable by exposing the polypeptide,e.g., an unglycosylated form, to appropriate glycosylating proteinsintroduced into a heterologous expression system. For example, theinterleukin gene may be co-transformed with one or more genes encodingmammalian or other glycosylating enzymes. Using this approach, certainmammalian glycosylation patterns will be achievable in prokaryote orother cells.

The source of IL-1ζ can be a eukaryotic or prokaryotic host expressingrecombinant IL-1ζ DNA, such as is described above. The source can alsobe a cell line such as mouse Swiss 3T3 fibroblasts, but other mammaliancell lines are also contemplated by this invention, with the preferredcell line being from the human species.

Now that the entire sequence is known, the primate (human) IL-1ζ,fragments, or derivatives thereof can be prepared by conventionalprocesses for synthesizing peptides. These include processes such as aredescribed in Stewart and Young (1984) Solid Phase Peptide Synthesis,Pierce Chemical Co., Rockford, Ill.; Bodanszky and Bodanszky (1984) ThePractice of Peptide Synthesis, Springer-Verlag, New York; and Bodanszky(1984) The Principles of Peptide Synthesis, Springer-Verlag, New York;all of each which are incorporated herein by reference. For example, anazide process, an acid chloride process, an acid anhydride process, amixed anhydride process, an active ester process (e.g., p-nitrophenylester, N-hydroxysuccinimide ester, or cyanomethyl ester), acarbodiimidazole process, an oxidative-reductive process, or adicyclohexylcarbodiimide (DCCD)/additive process can be used. Solidphase and solution phase syntheses are both applicable to the foregoingprocesses.

The IL-1ζ protein, fragments, or derivatives are suitably prepared inaccordance with the above processes as typically employed in peptidesynthesis, generally either by a so-called stepwise process whichcomprises condensing an amino acid to the terminal amino acid, one byone in sequence, or by coupling peptide fragments to the terminal aminoacid. Amino groups that are not being used in the coupling reactiontypically must be protected to prevent coupling at an incorrectlocation.

If a solid phase synthesis is adopted, the C-terminal amino acid isbound to an insoluble carrier or support through its carboxyl group. Theinsoluble carrier is not particularly limited as long as it has abinding capability to a reactive carboxyl group. Examples of suchinsoluble carriers include halomethyl resins, such as chloromethyl resinor bromomethyl resin, hydroxymethyl resins, phenol resins,tert-alkyloxycarbonylhydrazidated resins, and the like.

An amino group-protected amino acid is bound in sequence throughcondensation of its activated carboxyl group and the reactive aminogroup of the previously formed peptide or chain, to synthesize thepeptide step by step. After synthesizing the complete sequence, thepeptide is split off from the insoluble carrier to produce the peptide.This solid-phase approach is generally described by Merrifield, et al.(1963) in J. Am. Chem. Soc. 85:2149-2156, which is incorporated hereinby reference.

The prepared protein and fragments thereof can be isolated and purifiedfrom the reaction mixture by means of peptide separation, e.g., byextraction, precipitation, electrophoresis, various forms ofchromatography, and the like. The interleukin of this invention can beobtained in varying degrees of purity depending upon its desired use.Purification can be accomplished by use of the protein purificationtechniques disclosed herein, see below, or by the use of the antibodiesherein described in methods of immunoabsorbant affinity chromatography.This immunoabsorbant affinity chromatography is carried out by firstlinking the antibodies to a solid support and then contacting the linkedantibodies with solubilized lysates of appropriate cells, lysates ofother cells expressing the interleukin, or lysates or supernatants ofcells producing the protein as a result of DNA techniques, see below.

Generally, the purified protein will be at least about 40% pure,ordinarily at least about 50% pure, usually at least about 60% pure,typically at least about 70% pure, more typically at least about 80%pure, preferable at least about 90% pure and more preferably at leastabout 95% pure, and in particular embodiments, 97%-99% or more. Puritywill usually be on a weight basis, but can also be on a molar basis.Different assays will be applied as appropriate.

VI. Antibodies

The term “antibody” or “antibody molecule” as used in this inventionincludes intact molecules as well as fragments thereof, such as Fab,F(ab′)₂, and Fv which are capable of binding the epitopic determinant.These antibody fragments retain some ability to selectively bind withits antigen or receptor and are defined as follows: (1) Fab, thefragment which contains a monovalent antigen-binding fragment of anantibody molecule can be produced by digestion of whole IgG antibodywith the enzyme papain to yield an intact light chain and a portion ofone heavy chain; (2) Fab′, the fragment of an antibody molecule can beobtained by treating whole IgG antibody with pepsin, followed byreduction, to yield an intact light chain and a portion of the heavychain; two Fab′ fragments are obtained per antibody molecule; (3)(Fab′)₂, the fragment of the IgG antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds; (4) Fv, defined as a genetically engineered fragmentcontaining essentially the variable region of the light chain and thevariable region of the heavy chain expressed as two chains; and (5)Single Chain Antibody (“SCA”), defined as a genetically engineeredmolecule containing essentially the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

Methods of making these fragments are known in the art. See, e.g.,Harlow and Lane (current edition) Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York. Therefore, the phrase “antibodymolecule” in its various forms as used herein contemplates both anintact antibody (immunoglobulin) molecule and an immunologically activeportion of an antibody (immunoglobulin) molecule. Recombinant methodsmay be applied to make these fragments.

The term “monoclonal antibody” refers to a population of one species ofantibody molecule of antigen-specificity. A monoclonal antibody containsone species of antibody combining site capable of immunoreacting with aparticular antigen and thus typically displays a single binding affinityfor that antigen. A monoclonal antibody may therefore contain abispecific antibody molecule having two antibody combining sites, eachimmunospecific for a different antigen. In one embodiment, the firstantibody molecule is affixed to a solid support. In addition, theantibody molecules in a phage display combinatorial library are alsomonoclonal antibodies.

As used in this invention, the term “epitope” means an antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

The word “complex” as used herein refers to the product of a specificbinding agent-ligand reaction. An exemplary complex is an immunoreactionproduct formed by an antibody-antigen reaction.

The term “antigen” refers to a polypeptide or protein that is able toselectively bind to (immunoreact with) an antibody and form animmunoreaction product (immunocomplex). The site on the antigen to whichthe antibody binds is referred to as an antigenic determinant orepitope, and the labeling should be detectable, e.g., 2×, 5×, or moreabove background.

The method of the invention for detection of antibodies that bind tonovel epitopes in a sample is performed in vitro, e.g., in immunoassaysin which the antibodies can be identified in liquid phase or bound to asolid phase carrier. Preferably, the method is performed with a captureantibody bound to a solid support. Preferably, the capture antibody is amonoclonal antibody molecule.

Examples of types of immunoassays which can be utilized to detect novelantibodies in a sample, include competitive and non-competitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the radioimmunoassay (RIA) and the sandwich(immunometric) assay. Detection of the antibodies can be done utilizingimmunoassays which are run in either the forward, reverse, orsimultaneous modes, including competition immunoassays andimmunohistochemical assays on physiological samples. Preferably, themethod of the invention utilizes a forward immunoassay. Those of skillin the art will know, or can readily discern, other immunoassay formatswithout undue experimentation.

Solid phase-bound antibody molecules are bound by adsorption from anaqueous medium, although other modes of affixation, such as covalentcoupling or other well known means of affixation to the solid matrix canbe used. Preferably, the first antibody molecule is bound to a supportbefore forming an immunocomplex with antigen, however, the immunocomplexcan be formed prior to binding the complex to the solid support.

Non-specific protein binding sites on the surface of the solid phasesupport are preferably blocked. After adsorption of solid phase-boundantibodies, an aqueous solution of a protein free from interference withthe assay such as bovine, horse, or other serum albumin that is alsofree from contamination with the antigen is admixed with the solid phaseto adsorb the admixed protein onto the surface of theantibody-containing solid support at protein binding sites on thesurface that are not occupied by the antibody molecule.

A typical aqueous protein solution contains about 2-10 weight percentbovine serum albumin in PBS at a pH of about 7-8. The aqueous proteinsolution-solid support mixture is typically maintained for a time periodof at least one hour at a temperature of about 4°-37° C. and theresulting solid phase is thereafter rinsed free of unbound protein.

The first preselected antibody can be bound to many different carriersand used to detect novel epitope binding antibodies in a sample.Examples of well-known carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amyloses, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Thenature of the carrier can be either soluble or insoluble for purposes ofthe invention. Those skilled in the art will know of other suitablecarriers for binding antibodies, or will be able to ascertain such,using routine experimentation.

In addition, if desirable, an antibody for detection in theseimmunoassays can be detectably labeled in various ways. There are manydifferent labels and methods of labeling known to those of ordinaryskill in the art. Examples of the types of labels which can be used inthe present invention include enzymes, radioisotopes, fluorescentcompounds, colloidal metals, chemiluminescent compounds, andbio-luminescent compounds. Those of ordinary skill in the art will knowof other suitable labels for binding to the monoclonal antibodies of theinvention, or will be able to ascertain such, using routineexperimentation. Furthermore, the binding of these labels to theantibodies used in the method of the invention can be done usingstandard techniques common to those of ordinary skill in the art.

Antibodies which bind to IL-1ζ polypeptides of the invention can beprepared using an intact polypeptide or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide or apeptide used to immunize an animal can be derived from translated cDNAor chemical synthesis which can be conjugated to a carrier protein, ifdesired. Such commonly used carriers which are chemically coupled to thepeptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovineserum albumin (BSA), and tetanus toxoid. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

If desired, polyclonal or monoclonal antibodies can be further purified,e.g., by binding to and elution from a matrix to which the polypeptideor a peptide to which the antibodies were raised is bound. Those ofskill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies. See, e.g., Coligan, et al.(current ed.) Current Protocols in Immunology, Wiley Interscience.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, e.g., Green, et al. “Production of Polyclonal Antisera”pages 1-5 in Manson (ed.) Immunochemical Protocols Humana Press; Harlowand Lane; and Coligan, et al. Current Protocols in Immunology.

The preparation of monoclonal antibodies likewise is conventional. See,e.g., Kohler and Milstein (1975) Nature 256:495-497; Coligan, et al.,sections 2.5.1-2.6.7; and Harlow and Lane Antibodies: A LaboratoryManual, Cold Spring Harbor Press. Briefly, monoclonal antibodies can beobtained by injecting mice with a composition comprising an antigen,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B lymphocytes, fusing the Blymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones that produce antibodies to theantigen, and isolating the antibodies from the hybridoma cultures.Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See,e.g., Coligan, et al.; Barnes, et al. “Purification of Immunoglobulin G(IgG)” in Methods in Molecular Biology, vol. 10, pages 79-104 (HumanaPress, current ed.). Methods of in vitro and in vivo multiplication ofmonoclonal antibodies are well-known to those skilled in the art.Multiplication in vitro may be carried out in suitable culture mediasuch as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionallyreplenished, e.g., by a mammalian serum such as fetal calf serum ortrace elements and growth-sustaining supplements such as normal mouseperitoneal exudate cells, spleen cells, bone marrow macrophages.Production in vitro provides relatively pure antibody preparations andallows scale-up to yield large amounts of the desired antibodies. Largescale hybridoma cultivation can be carried out by homogenous suspensionculture in an airlift reactor, in a continuous stirrer reactor, or inimmobilized or entrapped cell culture. Multiplication in vivo may becarried out by injecting cell clones into mammals histocompatible withthe parent cells, e.g., syngeneic mice, to cause growth ofantibody-producing tumors. Optionally, the animals are primed with ahydrocarbon, especially oils such as pristane (tetramethylpentadecane)prior to injection. After one to three weeks, the desired monoclonalantibody is recovered from the body fluid of the animal.

Therapeutic applications are conceivable for the antibodies of thepresent invention. For example, antibodies of the present invention mayalso be derived from subhuman primate antibody. General techniques forraising therapeutically useful antibodies in baboons may be found, e.g.,in Goldenberg, et al. (1991) WO 91/11465; and Losman, et al. (1990) Int.J. Cancer 46:310-314.

Alternatively, a therapeutically useful anti-IL-1ζ antibody may bederived from a “humanized” monoclonal antibody. Humanized monoclonalantibodies are produced by transferring mouse complementary determiningregions from heavy and light variable chains of the mouse immunoglobulininto a human variable domain, and then substituting human residues inthe framework regions of the murine counterparts. The use of antibodycomponents derived from humanized monoclonal antibodies obviatespotential problems associated with the immunogenicity of murine constantregions. General techniques for cloning murine immunoglobulin variabledomains are described, e.g., by Orlandi, et al. (1989) Proc. Nat'l Acad.Sci. USA 86:3833-3837. Techniques for producing humanized monoclonalantibodies are described, e.g., by Jones et al. (1986) Nature321:522-525; Riechmann, et al. (1988) Nature 332:323-327; Verhoeyen, etal. (1988) Science 239:1534-1536; Carter, et al. (1992) Proc. Nat'lAcad. Sci. USA 89:4285-4289; Sandhu (1992) Crit. Rev. Biotech.12:437-462; and Singer, et al. (1993) J. Immunol. 150:2844-2857.

Antibodies of the invention also may be derived from human antibodyfragments isolated from a combinatorial immunoglobulin library. See,e.g., Barbas, et al. (1991) Methods: A Companion to Methods inEnzymology, vol. 2, page 119; and Winter, et al. (1994) Ann. Rev.Immunol. 12:433-455. Cloning and expression vectors that are useful forproducing a human immunoglobulin phage library can be obtained, e.g.,from STRATAGENE Cloning Systems (La Jolla, Calif.).

In addition, antibodies of the present invention may be derived from ahuman monoclonal antibody. Such antibodies are obtained from transgenicmice that have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Green,et al. (1994) Nature Genet. 7:13; Lonberg, et al. (1994) Nature 368:856;and Taylor, et al. (1994) Int. Immunol. 6:579.

Antibody fragments of the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ofDNA encoding the fragment. Antibody fragments can be obtained by pepsinor papain digestion of whole antibodies by conventional methods. Forexample, antibody fragments can be produced by enzymatic cleavage of IgGantibodies with pepsin to provide a 5S fragment denoted F(ab′)₂. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5 S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using papain producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, e.g., by Goldenberg, U.S. Pat. No. 4,036,945 and No.4,331,647, and references contained therein. These patents are herebyincorporated in their entireties by reference including all figures,drawings, and illustrations. See also Nisonhoff, et al. (1960) Arch.Biochem. Biophys. 89:230-xxx; Porter (1959) Biochem. J. 73:119-xxx;Edelman, et al. (1967) Methods in Enzymology, vol. 1, Academic Press;and Coligan, et al.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association may be noncovalent, as described in Inbar, etal. (1972) Proc. Nat'l Acad. Sci. USA 69:2659. Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu(1992) Crit. Rev. Biotech. 12:437. Preferably, the Fv fragments compriseV_(H) and V_(L) chains connected by a peptide linker. These single-chainantigen binding proteins (sFv) are prepared by constructing a structuralgene comprising DNA sequences encoding the V_(H) and V_(L) domainsconnected by an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, e.g., by Whitlow, et al.(1991) Methods: A Companion to Methods in Enzymology, vol. 2, page 97;Bird, et al. (1988) Science 242:423-426; Ladner, et al., U.S. Pat. No.4,946,778; Pack, et al. (1993) Bio/Technology 11:1271-77; and Sandhu(1992) Crit. Rev. Biotech. 12:437-462.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, e.g., by usingthe polymerase chain reaction to synthesize the variable region from RNAof antibody-producing cells. See, e.g., Larrick, et al. (1991) Methods:A Companion to Methods in Enzymology, vol. 2, page 106.

Antibodies can be raised to the various mammalian, e.g., primate IL-1ζpolypeptides, both in naturally occurring native forms and in theirdenatured forms, the difference being that antibodies to the activeligand are more likely to recognize epitopes which are only present inthe native conformations. Denatured antigen detection can also be usefulin, e.g., Western analysis. Anti-idiotypic antibodies are alsocontemplated, which would be useful as agonists or antagonists of anatural receptor or an antibody.

A number of immunogens may be used to produce antibodies selectivelyreactive with IL-1ζ proteins. Recombinant protein is the preferredimmunogen for the production of monoclonal or polyclonal antibodies.Naturally occurring protein may also be used either in pure or impureform. Synthetic peptides made using the IL-1ζ protein sequencesdescribed herein may also used as an immunogen for the production ofantibodies to the antigens. Recombinant protein can be expressed ineukaryotic or prokaryotic cells as described herein, and purified asdescribed. The product is then injected into an animal capable ofproducing antibodies. Either monoclonal or polyclonal antibodies may begenerated for subsequent use in immunoassays to measure the protein.

Methods of producing polyclonal antibodies are known to those of skillin the art. In brief, an immunogen, preferably a purified protein, ismixed with an adjuvant and animals are immunized with the mixture. Theanimal's immune response to the immunogen preparation is monitored bytaking test bleeds and determining the titer of reactivity to theprotein antigen of interest. When appropriately high titers of antibodyto the immunogen are obtained, blood is collected from the animal andantisera are prepared. Further fractionation of the antisera to enrichfor antibodies reactive to the protein can be done if desired. SeeHarlow and Lane.

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell. Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse, et al. (1989) Science 246:1275-1281.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of the protein can be raised byimmunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective protein, or screened for agonistic orantagonistic activity. These monoclonal antibodies will usually bindwith at least a K_(D) of about 1 mM, more usually at least about 300 μM,typically at least about 100 μM, more typically at least about 30 μM,preferably at least about 10 μM, and more preferably at least about 3 μMor better; including 1 μM, 300 nM, 100 nM, 30 nM, etc.

The antibodies, including antigen binding fragments, of this inventioncan have significant diagnostic or therapeutic value. They can be potentantagonists that bind to the interleukin and inhibit binding to thereceptor or inhibit the ability of IL-1ζ to elicit a biologicalresponse. They also can be useful as non-neutralizing antibodies and canbe coupled to toxins or radionuclides to bind producing cells, or cellslocalized to the source of the interleukin. Further, these antibodiescan be conjugated to drugs or other therapeutic agents, either directlyor indirectly by means of a linker.

The antibodies of this invention can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bindto the interleukin without inhibiting receptor binding. As neutralizingantibodies, they can be useful in competitive binding assays. They willalso be useful in detecting or quantifying IL-1ζ. They may be used asreagents for Western blot analysis, or for immunoprecipitation orimmunopurification of the respective protein.

Protein fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. Mammalian IL-1ζ and its fragments may be fused or covalentlylinked to a variety of immunogens, such as keyhole limpet hemocyanin,bovine serum albumin, tetanus toxoid, etc. See Microbiology, HoeberMedical Division, Harper and Row, 1969; Landsteiner (1962) Specificityof Serological Reactions, Dover Publications, New York; and Williams, etal. (1967) Methods in Immunology and Immunochemistry, Vol. 1, AcademicPress, New York; each of which are incorporated herein by reference, fordescriptions of methods of preparing polyclonal antisera. A typicalmethod involves hyperimmunization of an animal with an antigen. Theblood of the animal is then collected shortly after the repeatedimmunizations and the gamma globulin is isolated.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology(4th ed.), Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual,CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.) Academic Press, New York; and particularly in Kohler andMilstein (1975) in Nature 256: 495-497, which discusses one method ofgenerating monoclonal antibodies. Each of these references isincorporated herein by reference. Summarized briefly, this methodinvolves injecting an animal with an immunogen. The animal is thensacrificed and cells taken from its spleen, which are then fused withmyeloma cells. The result is a hybrid cell or “hybridoma” that iscapable of reproducing in vitro. The population of hybridomas is thenscreened to isolate individual clones, each of which secrete a singleantibody species to the immunogen. In this manner, the individualantibody species obtained are the products of immortalized and clonedsingle B cells from the immune animal generated in response to aspecific site recognized on the immunogenic substance.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse, et al. (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246:1275-1281; and Ward, et al.(1989) Nature 341:544-546, each of which is hereby incorporated hereinby reference. The polypeptides and antibodies of the present inventionmay be used with or without modification, including chimeric orhumanized antibodies. Frequently, the polypeptides and antibodies willbe labeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmoieties, chemiluminescent moieties, magnetic particles, and the like.Patents, teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant or chimeric immunoglobulins may beproduced, see Cabilly, U.S. Pat. No. 4,816,567; or made in transgenicmice, see Mendez, et al. (1997) Nature Genetics 15:146-156. Thesereferences are incorporated herein by reference.

The antibodies of this invention can also be used for affinitychromatography in isolating the IL-1ζ. Columns can be prepared where theantibodies are linked to a solid support, e.g., particles, such asagarose, SEPHADEX, or the like, where a cell lysate may be passedthrough the column, the column washed, followed by increasingconcentrations of a mild denaturant, whereby the purified protein willbe released. The protein may be used to purify antibody.

The antibodies may also be used to screen expression libraries forparticular expression products. Usually the antibodies used in such aprocedure will be labeled with a moiety allowing easy detection ofpresence of antigen by antibody binding.

Antibodies raised against an IL-1ζ will also be used to raiseanti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of theprotein or cells which express receptors for the protein. They also willbe useful as agonists or antagonists of the interleukin, which may becompetitive inhibitors or substitutes for naturally occurring ligands.

Binding Agent:IL-1ζ Polypeptide Complex

An IL-1ζ polypeptide that specifically binds to or that is specificallyimmunoreactive with an antibody, e.g., such as a polyclonal antibody,generated against a defined immunogen, e.g., such as an immunogenconsisting of an amino acid sequence of SEQ ID NO: 2 or fragmentsthereof or a polypeptide generated from the nucleic acid of SEQ ID NO: 1is typically determined in an immunoassay. Included within the metes andbounds of the present invention are those nucleic acid sequencesdescribed herein, including functional variants, that encodepolypeptides that selectively bind to polyclonal antibodies generatedagainst the prototypical IL-1ζ polypeptide as structurally andfunctionally defined herein. The immunoassay typically uses a polyclonalantiserum which was raised, e.g., to a protein of SEQ ID NO: 2. Thisantiserum is selected to have low crossreactivity against other IL-1family members, preferably from the same species, and suchcrossreactivity is removed by immunoabsorption or depletion prior to usein the immunoassay. Appropriate selective serum preparations can beisolated, and characterized.

In order to produce antisera for use in an immunoassay, the protein ofSEQ ID NO: 2 is isolated as described herein. For example, recombinantprotein may be produced in a mammalian cell line. An appropriate host,e.g., an inbred strain of mice such as Balb/c, is immunized with theprotein of SEQ ID NO: 2 using a standard adjuvant, such as Freund'sadjuvant, and a standard mouse immunization protocol (see Harlow andLane). Alternatively, a synthetic peptide derived from the sequencesdisclosed herein and conjugated to a carrier protein can be used as animmunogen. Polyclonal sera are collected and titered against theimmunogen protein in an immunoassay, e.g., a solid phase immunoassaywith the immunogen immobilized on a solid support. Polyclonal antiserawith a titer of 10⁴ or greater are selected and tested for their crossreactivity against other IL-1 family members, e.g., IL-1α, IL-1β,IL-1RA, IL-1γ, IL-1δ, and IL-1ε, using a competitive binding immunoassaysuch as the one described in Harlow and Lane, supra, at pages 570-573.Preferably at least two IL-1 family members are used in thisdetermination in conjunction with IL-1ζ. These IL-1 family members canbe produced as recombinant proteins and isolated using standardmolecular biology and protein chemistry techniques as described herein.Thus, antibody preparations can be identified or produced having desiredselectivity or specificity for subsets of IL-1 family members.

Immunoassays in the competitive binding format can be used for thecrossreactivity determinations. For example, the protein of SEQ ID NO: 2can be immobilized to a solid support. Proteins added to the assaycompete with the binding of the antisera to the immobilized antigen. Theability of the above proteins to compete with the binding of theantisera to the immobilized protein is compared to the protein of SEQ IDNO: 2. The percent crossreactivity for the above proteins is calculated,using standard calculations. Those antisera with less than 10%crossreactivity with each of the proteins listed above are selected andpooled. The cross-reacting antibodies are then removed from the pooledantisera by immunoabsorption with the above-listed proteins.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein tothe immunogen protein. In order to make this comparison, the twoproteins are each assayed at a wide range of concentrations and theamount of each protein required to inhibit 50% of the binding of theantisera to the immobilized protein is determined. If the amount of thesecond protein required is less than twice the amount of the protein ofSEQ ID NO: 2 that is required, then the second protein is said tospecifically bind to an antibody generated to the immunogen.

It is understood that this IL-1ζ polypeptide is a member of a family ofhomologous proteins that comprise at least 6 so far identified genes.For a particular gene product, such as the IL-1ζ, the term refers notonly to the amino acid sequences disclosed herein, but also to otherproteins that are allelic, non-allelic or species variants. It alsounderstood that the term “IL-1ζ” includes nonnatural mutationsintroduced by deliberate mutation using conventional recombinanttechnology such as single site mutation, or by excising short sectionsof DNA encoding the respective proteins, or by substituting new aminoacids, or adding new amino acids. Such minor alterations mustsubstantially maintain the immunoidentity of the original moleculeand/or its biological activity. Thus, these alterations include proteinsthat are specifically immunoreactive with a designated naturallyoccurring IL-1 related protein, e.g., the IL-1ζ polypeptide shown in SEQID NO: 2. The biological properties of the altered proteins can bedetermined by expressing the protein in an appropriate cell line andmeasuring the appropriate effect upon lymphocytes. Particular proteinmodifications considered minor would include conservative substitutionof amino acids with similar chemical properties, as described above forthe IL-1 family as a whole. By aligning a protein optimally with theprotein of SEQ ID NO: 2 and by using the conventional immunoassaysdescribed herein to determine immunoidentity, one can determine theprotein compositions of the invention.

VII. Kits and Quantitation

Both naturally occurring and recombinant forms of the IL-1 likemolecules of this invention are particularly useful in kits and assaymethods. For example, these methods would also be applied to screeningfor binding activity, e.g., receptors for these proteins. Severalmethods of automating assays have been developed in recent years so asto permit screening of tens of thousands of compounds per year. See,e.g., a BIOMEK automated workstation, Beckman Instruments, Palo Alto,Calif., and Fodor, et al. (1991) Science 251:767-773, which isincorporated herein by reference. The latter describes means for testingbinding by a plurality of defined polymers synthesized on a solidsubstrate. The development of suitable assays to screen for a receptoror agonist/antagonist homologous proteins can be greatly facilitated bythe availability of large amounts of purified, soluble IL-1ζ in anactive state such as is provided by this invention.

Purified IL-1ζ can be coated directly onto plates for use in theaforementioned receptor screening techniques. However, non-neutralizingantibodies to these proteins can be used as capture antibodies toimmobilize the respective interleukin on the solid phase, useful, e.g.,in diagnostic uses.

This invention also contemplates use of IL-1ζ polypeptides and theirfusion products in a variety of diagnostic kits and methods fordetecting the presence of the protein or its receptor. Alternatively, oradditionally, antibodies against the molecules may be incorporated intothe kits and methods. Typically the kit will have a compartmentcontaining either a defined IL-1ζ peptide or gene segment or a reagentwhich recognizes one or the other. Typically, recognition reagents, inthe case of peptide, would be a receptor or antibody, or in the case ofa gene segment, would usually be a hybridization probe.

A preferred kit for determining the concentration of, e.g., IL-1ζ, asample would typically comprise a labeled compound, e.g., receptor orantibody, having known binding affinity for IL-1ζ, a source of IL-1ζ(naturally occurring or recombinant) as a positive control, and a meansfor separating the bound from free labeled compound, e.g., a solid phasefor immobilizing the IL-1ζ in the test sample. Compartments containingreagents, and instructions, will normally be provided.

Antibodies, including antigen binding fragments, specific for mammalianIL-1ζ or a peptide fragment, or receptor fragments are useful indiagnostic applications to detect the presence of elevated levels ofIL-1ζ and/or its fragments. Diagnostic assays may be homogeneous(without a separation step between free reagent and antibody-antigencomplex) or heterogeneous (with a separation step). Various commercialassays exist, such as radioimmunoassay (RIA), enzyme-linkedimmunosorbent assay (ELISA), enzyme immunoassay (EIA), enzyme-multipliedimmunoassay technique (EMIT), substrate-labeled fluorescent immunoassay(SLFIA) and the like. For example, unlabeled antibodies can be employedby using a second antibody which is labeled and which recognizes theantibody to IL-1ζ or to a particular fragment thereof. These assays havealso been extensively discussed in the literature. See, e.g., Harlow andLane (1988) Antibodies: A Laboratory Manual, CSH., and Coligan (ed. 1991and periodic supplements) Current Protocols In Immunology Greene/Wiley,New York.

Anti-idiotypic antibodies may have similar use to serve as agonists orantagonists of IL-1ζ. These should be useful as therapeutic reagentsunder appropriate circumstances.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody, or labeled receptor is provided.This is usually in conjunction with other additives, such as buffers,stabilizers, materials necessary for signal production such assubstrates for enzymes, and the like. Preferably, the kit will alsocontain instructions for proper use and disposal of the contents afteruse. Typically the kit has compartments for each useful reagent, andwill contain instructions for proper use and disposal of reagents.Desirably, the reagents are provided as a dry lyophilized powder, wherethe reagents may be reconstituted in an aqueous medium havingappropriate concentrations for performing the assay.

The aforementioned constituents of the diagnostic assays may be usedwithout modification or may be modified in a variety of ways. Forexample, labeling may be achieved by covalently or non-covalentlyjoining a moiety which directly or indirectly provides a detectablesignal. In these assays, a test compound, IL-1ζ, or antibodies theretocan be labeled either directly or indirectly. Possibilities for directlabeling include label groups: radiolabels such as ¹²⁵I, enzymes (U.S.Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase, andfluorescent labels (U.S. Pat. No. 3,940,475) capable of monitoring thechange in fluorescence intensity, wavelength shift, or fluorescencepolarization. Both of the patents are incorporated herein by reference.Possibilities for indirect labeling include biotinylation of oneconstituent followed by binding to avidin coupled to one of the abovelabel groups.

There are also numerous methods of separating the bound from the freeligand, or alternatively the bound from the free test compound. TheIL-1ζ can be immobilized on various matrixes followed by washing.Suitable matrices include plastic such as an ELISA plate, filters, andbeads. Methods of immobilizing the receptor to a matrix include, withoutlimitation, direct adhesion to plastic, use of a capture antibody,chemical coupling, and biotin-avidin. The last step in this approachinvolves the precipitation of antibody/antigen complex by appropriatemethods including those utilizing, e.g., an organic solvent such aspolyethylene glycol or a salt such as ammonium sulfate. Other suitableseparation techniques include, without limitation, the fluoresceinantibody magnetizable particle method described in Rattle, et al. (1984)Clin. Chem. 30:1457-1461, and the double antibody magnetic particleseparation as described in U.S. Pat. No. 4,659,678, each of which isincorporated herein by reference.

The methods for linking protein or fragments to various labels have beenextensively reported in the literature and do not require detaileddiscussion here. Many of the techniques involve the use of activatedcarboxyl groups either through the use of carbodiimide or active estersto form peptide bonds, the formation of thioethers by reaction of amercapto group with an activated halogen such as chloroacetyl, or anactivated olefin such as maleimide, for linkage, or the like. Fusionproteins will also find use in these applications.

Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence ofan IL-1ζ. These sequences can be used as probes for detecting levels ofthe IL-1ζ in patients suspected of having an immunological disorder. Thepreparation of both RNA and DNA nucleotide sequences, the labeling ofthe sequences, and the preferred size of the sequences has receivedample description and discussion in the literature. Normally anoligonucleotide probe should have at least about 14 nucleotides, usuallyat least about 18 nucleotides, and the polynucleotide probes may be upto several kilobases. Various labels may be employed, most commonlyradionuclides, particularly ³²P. However, other techniques may also beemployed, such as using biotin modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed which can recognize specificduplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes,or DNA-protein duplexes. The antibodies in turn may be labeled and theassay carried out where the duplex is bound to a surface, so that uponthe formation of duplex on the surface, the presence of antibody boundto the duplex can be detected. The use of probes to the novel anti-senseRNA may be carried out in conventional techniques such as nucleic acidhybridization, plus and minus screening, recombinational probing, hybridreleased translation (HRT), and hybrid arrested translation (HART). Thisalso includes amplification techniques such as polymerase chain reaction(PCR).

Diagnostic kits which also test for the qualitative or quantitativepresence of other markers are also contemplated. Diagnosis or prognosismay depend on the combination of multiple indications used as markers.Thus, kits may test for combinations of markers. See, e.g., Viallet, etal. (1989) Progress in Growth Factor Res. 1:89-97.

VIII. Therapeutic Utility

This invention provides reagents with significant therapeutic value. TheIL-1ζ polypeptides (naturally occurring or recombinant), mutein agonistsand antagonists, and antibodies, along with compounds identified ashaving binding affinity to the interleukin or its receptor orantibodies, should be useful in the treatment of conditions exhibitingabnormal expression of the interleukin. Such abnormality will typicallybe manifested by immunological disorders. Additionally, this inventionshould provide therapeutic value in various diseases or disordersassociated with abnormal expression or abnormal triggering of responseto the interleukin. The mouse IL-γ has been suggested to be involved intumors, allergies, and infectious diseases, e.g., pulmonarytuberculosis, leprosy, fulminant hepatitis, and viral infections, suchas HIV. The IL-1ζ or antagonist may have similar function, suggestingcombination compositions with other agonists or antagonists of IL-1family members.

T helper cells mediate effector functions in infectious, allergic, orautoimmune diseases through production of cytokines. CD4 positive Tcells can be divided into Th1 and Th2 subsets on the basis of theircytokine profile upon antigen stimulation. Recently obtained evidencehas shown that Th1 and Th2 cells differ in responsiveness and receptorexpression for IL-1 family molecules. See, e.g., Robinson, et al. (1997)Immunity 7:571-581. Whereas Th1 cells respond to IL-1γ, Th2 cellsrespond to IL-1α. This differential responsiveness between Th1 and Th2cells to IL-1γ and IL-1α, respectively, may have profound implicationsfor regulation of ongoing Th cell responses. The novel IL-1 moleculesdescribed here could play a similar role in either supporting a Th1 orTh2 response, depending on the presence or absence of their cognate IL-1receptors on the cell surface of these immune cells; e.g., IL-1RD4 (ST2)is an orphan IL-1-like receptor exclusively expressed on the Th2 subset.See, e.g., Lohning, et al. (1998) Proc. Nat'l Acad. Sci. USA95:6930-6935; and U.S. Ser. No. 09/040,714, which are incorporatedherein by reference.

In addition, the dendritic cell expression profile shows human IL-1γprimarily expressed in activated dendritic cells. Activated dendriticcells are also a major producer of IL-12, and it is thought that thisdendritic cell produced IL-12 plays a major role in directing a Th1 typeresponse. The combination of IL-1γ and IL-12 should be extremely potentin inducing IFN-γ, suggesting that IL-1ζ, or antagonists thereof, mayhave similar function. It is possible that the combination ofpro-inflammatory cytokines under certain circumstances could lead toseptic shock. An antagonist, mutein or antibody, could prove very usefulin this situation. See Rich (ed.) Clinical Immunology: Principles andPractice, Mosby.

Additionally, IL-1ζ being homologous members of the IL-1 family likelyplay a role in modulating of local and systemic inflammatory processes(see, Durum, et al. (1986) Ann. Rev. Immunol. 3:253-xxx), through theenhancement of blood flow, induction of chemoattractants, and theenhancement and adherence of adhesion molecules resulting in theaccumulation of inflammatory cells such as macrophages and neutrophilsat the site of inflammation. Additionally, it is possible that IL-1ζ caninduce fibroblast growth and may play a role in contributing to thepathogenesis of chronic inflammation, as in rheumatoid arthritis orperiodontal disease.

IL-1ζ is also likely to play a role in systemic inflammatory reactions,such as fever, hypoglycemia, the acute phase response of the liver,reduced plasma iron and zinc, and increased plasma copper. A systemicreaction such as septic shock involves vasodilation, due to IL-1, mostlikely in combination with other cytokines, including, e.g., TNF, IFN-γ,and leukemia inhibitory factor (LIF). The newly described IL-1ζ is alsolikely to be similarly involved.

Recombinant IL-1ζ, mutein agonists or antagonists, or IL-1ζ antibodiescan be purified and then administered to a patient. These reagents canbe combined for therapeutic use with additional active ingredients,e.g., in conventional pharmaceutically acceptable carriers or diluents,along with physiologically innocuous stabilizers and excipients. Thesecombinations can be sterile, e.g., filtered, and placed into dosageforms as by lyophilization in dosage vials or storage in stabilizedaqueous preparations. This invention also contemplates use of antibodiesor binding fragments thereof which are not complement binding.

Receptor screening using IL-1ζ or fragments thereof can be performed toidentify molecules having binding affinity to the interleukin.Subsequent biological assays can then be utilized to determine if areceptor can provide competitive binding, which can block intrinsicstimulating activity. Receptor fragments can be used as a blocker orantagonist in that it blocks the activity of IL-1ζ. Likewise, a compoundhaving intrinsic stimulating activity can activate the receptor and isthus an agonist in that it simulates the activity of IL-1ζ. Thisinvention further contemplates the therapeutic use of antibodies toIL-1ζ as antagonists.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics,8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences(current ed.), Mack Publishing Co., Easton, Pa.; each of which is herebyincorporated herein by reference. Methods for administration arediscussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Because of the likely high affinitybinding between an IL-1ζ and its receptors, low dosages of thesereagents would be initially expected to be effective. And the signalingpathway suggests extremely low amounts of ligand may have effect. Thus,dosage ranges would ordinarily be expected to be in amounts lower than 1mM concentrations, typically less than about 10 μM concentrations,usually less than about 100 nM, preferably less than about 10 pM(picomolar), and most preferably less than about 1 fM (femtomolar), withan appropriate carrier. Slow release formulations, or slow releaseapparatus will often be utilized for continuous administration.

IL-1ζ polypeptides, and antibodies or its fragments, antagonists, andagonists, may be administered directly to the host to be treated or,depending on the size of the compounds, it may be desirable to conjugatethem to carrier proteins such as ovalbumin or serum albumin prior totheir administration. Therapeutic formulations may be administered in aconventional dosage formulation. While it is possible for the activeingredient to be administered alone, it is preferable to present it as apharmaceutical formulation. Formulations comprise at least one activeingredient, as defined above, together with one or more acceptablecarriers thereof. Each carrier must be both pharmaceutically andphysiologically acceptable in the sense of being compatible with theother ingredients and not injurious to the patient. Formulations includethose suitable for oral, rectal, nasal, or parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal)administration. The formulations may conveniently be presented in unitdosage form and may be prepared by methods well known in the art ofpharmacy. See, e.g., Gilman, et al. (eds. 1990) Goodman and Gilman's:The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; andRemington's Pharmaceutical Sciences, 17th ed. (1990), Mack PublishingCo., Easton, Pa.; Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms:Parenteral Medications Dekker, NY; Lieberman, et al. (eds. 1990)Pharmaceutical Dosage Forms: Tablets Dekker, NY; and Lieberman, et al.(eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, NY.

Another therapeutic approach included within the invention involvesdirect administration of reagents or compositions by conventionaladministration techniques (e.g., but not restricted to local injection,inhalation, or administered systemically), to the subject with aninflammatory disorder. The reagent, formulation or composition may alsobe targeted to specific cells or receptors by, e.g., methods describedherein. The actual dosage of reagent, formulation or composition thatmodulates an inflammatory disorder depends on many factors, includingthe size and health of an organism, however one of one of ordinary skillin the art can use the following teachings describing the methods andtechniques for determining clinical dosages. See, e.g., Spilker (1984)Guide to Clinical Studies and Developing Protocols, Raven Press, NewYork; Spilker (1991) Guide to Clinical Trials, Raven Press, New York;Craig and Stitzel (eds. 1986) Modern Pharmacology 2d ed., Little, Brown,Boston; Speight (ed. 1987) Avery's Drug Treatment: Principles andPractice of Clinical Pharmacology and Therapeutics, 3d ed., Williams andWilkins, Baltimore; and Tallarida, et al. (1988) Principles in GeneralPharmacology, Springer-Verlag, New York; which describe how to determinethe appropriate dosage; but, generally, in the range of about between0.5 ng/ml and 500 μg/ml inclusive final concentration are administeredper day to an adult in a pharmaceutically-acceptable carrier. Thetherapy of this invention may be combined with or used in associationwith other therapeutic agents, particularly agonists or antagonists ofother IL-1 family members.

IX. Receptors

The description of the IL-1ζ ligand herein provides means to identify areceptor, as described above. Such receptor should bind specifically tothe IL-1ζ with reasonably high affinity. Various constructs are madeavailable which allow either labeling of the IL-1ζ to detect itsreceptor. For example, directly labeling IL-1ζ, fusing onto it markersfor secondary labeling, e.g., FLAG or other epitope tags, etc., willallow detection of receptor. This can be histological, as an affinitymethod for biochemical purification, or labeling or selection in anexpression cloning approach. A two-hybrid selection system may also beapplied making appropriate constructs with the available IL-1ζsequences. See, e.g., Fields and Song (1989) Nature 340:245-246.Typically, a cytokine will bind to its receptor at a Kd of at leastabout 30 μM, preferably at least about 10 μM, and more preferably atleast about 3 μM or better; including 1 μM, 300 nM, 100 nM, 30 nM, etc.

Examples I. General Methods

Some of the standard methods are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols 1-3, CSHPress, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology, Greene/Wiley, New York. Methods forprotein purification include such methods as ammonium sulfateprecipitation, column chromatography, electrophoresis, centrifugation,crystallization, and others. See, e.g., Ausubel, et al. (1987 andperiodic supplements); Deutscher (1990) “Guide to Protein Purification”in Meth. Enzmmol., vol. 182, and other volumes in this series; andmanufacturer's literature on use of protein purification products, e.g.,Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, Calif. Combinationwith recombinant techniques allow fusion to appropriate segments, e.g.,to a FLAG sequence or an equivalent which can be fused via aprotease-removable sequence. See, e.g., Hochuli (1989) ChemischeIndustrie 12:69-70; Hochuli (1990) “Purification of Recombinant Proteinswith Metal Chelate Absorbent” in Setlow (ed.) Genetic Engineering,Principle and Methods 12:87-98, Plenum Press, N.Y.; and Crowe, et al.(1992) QIAexpress: The High Level Expression & Protein PurificationSystem QIAGEN, Inc., Chatsworth, Calif.

Computer sequence analysis is performed, e.g., using available softwareprograms, including those from the GCG (U. Wisconsin) and GenBanksources. Public sequence databases were also used, e.g., from GenBankand others.

Many techniques applicable to IL-4, IL-10, and IL-1γ, IL-1δ, and IL-1εmay be applied to IL-1ζ, as described, e.g., in U.S. Pat. No. 5,017,691(IL-4), U.S. Ser. No. 07/453,951 (IL-10), U.S. Ser. No. 08/110,683(IL-10 receptor); U.S. Ser. No. 08/651,998 (IL-1γ); U.S. Ser. No.09/062,866 or U.S. Ser. No. 09/097,976 (IL-1δ and IL-1ε), each of whichis incorporated herein by reference for all purposes.

II. Amplification of IL-1ζ Fragment by PCR

There are various methods of isolating the DNA sequences encoding IL-1ζpolypeptides. For example, DNA is isolated from a genomic or cDNAlibrary using labeled oligonucleotide probes having sequences identicalor complementary to the sequences disclosed herein. Full-length probesmay be used, or oligonucleotide probes may be generated by comparison ofthe sequences disclosed. Such probes can be used directly inhybridization assays to isolate DNA encoding IL-1ζ polypeptides, orprobes can be designed for use in amplification techniques such as PCR,for the isolation of DNA encoding IL-1ζ polypeptides.

Various methods of amplifying target sequences, such as the polymerasechain reaction, can also be used to prepare DNA encoding IL-1ζpolypeptides. Polymerase chain reaction (PCR) technology is used toamplify such nucleic acid sequences directly from mRNA, from cDNA, andfrom genomic libraries or cDNA libraries. The isolated sequencesencoding IL-1ζ polypeptides may also be used as templates for PCRamplification.

In PCR techniques, oligonucleotide primers complementary to two 5′regions in the DNA region to be amplified are synthesized. Thepolymerase chain reaction is then carried out using the two primers. SeeInnis, et al. (current eds.) PCR Protocols: A Guide to Methods andApplications Academic Press, San Diego, Calif. Primers can be selectedto amplify the entire regions encoding a full-length IL-1ζ polypeptideor to amplify smaller DNA segments, as desired. Once such regions arePCR-amplified, they can be sequenced and oligonucleotide probes can beprepared from sequence obtained using standard techniques. These probescan then be used to isolate DNA's encoding IL-1ζ polypeptides.

Oligonucleotides for use as probes are chemically synthesized, e.g.,according to the solid phase phosphoramidite triester method firstdescribed by Beaucage and Carruthers (1983) Tetrahedron Lett.22:1859-1862, or using an automated synthesizer, as described inNeedham-VanDevanter, et al. (1984) Nucleic Acids Res. 12:6159-6168.Purification of oligonucleotides is performed, e.g., by nativeacrylamide gel electrophoresis or by anion-exchange HPLC as described inPearson and Regnier (1983) J. Chrom. 255: 137-149. The sequence of thesynthetic oligonucleotide can be verified using the chemical degradationmethod of Maxam and Gilbert in Grossman and Moldave (eds. 1980) Methodsin Enzymology 65: 499-560 Academic Press, New York.

The peptide segments, along with comparison to homologous genes, canalso be used to produce appropriate oligonucleotides to screen alibrary. The genetic code can be used to select appropriateoligonucleotides useful as probes for screening. In combination withpolymerase chain reaction (PCR) techniques, synthetic oligonucleotideswill be useful in selecting desired clones from a library.

Complementary sequences will also be used as probes or primers. Basedupon identification of the likely amino terminus, other peptides shouldbe particularly useful, e.g., coupled with anchored vector or poly-Acomplementary PCR techniques or with complementary DNA of otherpeptides.

To identify a homologous IL-1ζ polypeptide, degenerate oligonucleotidesare designed which corresponded to conserved regions among known IL-1family members. The primers are used for polymerase chain reactions onprimate genomic DNA followed by subcloning the PCR products usingrestriction sites placed at the 5′ ends of the primers, pickingindividual E. coli colonies carrying these subcloned inserts, and usinga combination of random sequencing and hybridization analysis toeliminate known IL-1 family members.

Subsequently, PCR products are gel-purified, digested with appropriaterestriction enzymes, gel-purified again, and subcloned in the Bluescriptvector (Stratagene, San Diego, Calif.). Bacterial colonies carryingindividual subclones are picked into 96 well microtiter plates, andmultiple replicas are prepared by plating the cells onto nitrocellulose.The replicate filters are hybridized to probes representing knownmembers of the IL-1 family, and DNA is prepared from non-hybridizingcolonies for sequence analysis.

Two appropriate forward and reverse primers are selected using thesequences supplied herein (see Table 1) and common knowledge. See, e.g.,Innis, et al. (current eds.) PCR Protocols: A Guide to Methods andApplications Academic Press, San Diego, Calif.; and Dieffenbach andDveksler (current eds.) PCR Primer: A Laboratory Manual Cold SpringHarbor Press, CSH, NY. RT-PCR is used on an appropriate mRNA sampleselected for the presence of message to produce a cDNA, e.g., a monocyteor macrophage cell sample.

Full length clones may be isolated by hybridization of cDNA librariesfrom appropriate tissues pre-selected by PCR signal.

As is commonly known, PCR primers are typically designed to contain atleast 15 nucleotides, e.g., 15-30 nucleotides. The design of IL-1ζspecific primers containing 21 nucleotides, e.g., that code for IL-1ζpolypeptides containing at least 6 amino acids from the IL-1ζ domainsare described as follows. Other PCR primers designed to amplify otherIL-1ζ polypeptide fragments are designed in a similar fashion, e.g.,mutagenesis primers. Preferably, most or all of the nucleotides in sucha primer encode conserved amino acids, e.g., amino residues of SEQ. IDNO: 2 or 4, including IL-1ζ-specific amino acids as described herein.For example, primers containing at least 40% IL-1ζ-conserved amino acidscan be used. Such a primer, containing 21 nucleotides, can includesequences encoding at least 3/7, 4/7, 5/7, 6/7 or 7/7 IL-1ζ-conservedamino acids. Once IL-1ζ amino acids are selected as templates againstwhich primer sequences are to be designed, the primers can besynthesized using, e.g., standard chemical methods. Due to thedegeneracy of the genetic code and the bias of preferred speciesvariants, such primers should be designed to include appropriatedegenerate sequences, as can be readily determined using commonknowledge.

Based on the guidelines presented above, IL-1ζ-conserved amino acidpeptides can be used as templates for the design of IL-1ζ specificprimers. Additional examples can be found by analysis of sequencealignments of IL-1ζ polypeptides (Table 1 and 2). Primers can bedesigned to amplify various structural features or domains, e.g., a 4-10amino acid region of either IL-1ζ polypeptide that corresponds to one ofthe 12 β strands could be amplified using this strategy. Depending onthe length of the primer, desired primers can be designed, e.g., tocorrespond to 4-7 consecutive amino acids of the segments. Preferredsegments include, e.g., GENSGVK; EDWEKD; CCLEDPA; FVHTSR; KKFSIHD;VLVLDS; NLIAVP; FFALAS; SSASAEK; SLILLGV; FCLYCDK; PSLQLK; KLMKLAAQ;FIFYRAQ; SRNMLES; WFICTS; EPVGVT; or FSFQPVC (see SEQ ID NO: 2); orFVHTSP; SPILLGV; or SWNMLES (see SEQ ID NO: 4). Longer preferredsegments include, e.g., GVKMGSEDWEKD; AGSPLEPGPSLP; SRKVKSLNPKKF;HDQDHKVLVLDS; NLIAVPDKNYIR; FALASSLSSASA; GQSHPSLQLKKE; MKLAAQKESARR;FYRAQVGSRNML; TSCNCNEPVGVT; FENRKHIEFSFQ; or PVCKAEMSPSEV (see SEQ IDNO: 2); or AVSPLEPGPSLP; SPKVKNLNPKKF; or FYRAQVGSWNML (see SEQ ID NO:4).

As is described above, IL-1ζ primers, e.g., primers based on IL-1ζspecific peptides shown above, or portions thereof, can be used in PCRreactions to generate IL-1ζ probes which can be used in standardscreening methods to identify nucleic acids encoding IL-1 family members(see e.g., Ausubel, et al., supra).

III. Tissue Distribution of IL-1ζ

Message for the gene encoding IL-1ζ has been detected in a mixture offetal lung, testis, and B cell cDNAs. PCR has provided positive signalsin cDNA libraries derived from normal colon, lung, and a marginallydetectable signal from ovary. It was detectable by Northern blotting ina polyA+ sample from a colorectal adenocarcinoma.

Southern Analysis: DNA (5 μg) from a primary amplified cDNA library isdigested with appropriate restriction enzymes to release the inserts,run on a 1% agarose gel and transferred to a nylon membrane (Schleicherand Schuell, Keene, N.H.).

Samples for human mRNA isolation could include: peripheral bloodmononuclear cells (monocytes, T cells, NK cells, granulocytes, B cells),resting (T100); peripheral blood mononuclear cells, activated withanti-CD3 for 2, 6, 12 h pooled (T101); T cell, TH0 clone Mot 72, resting(T102); T cell, TH0 clone Mot 72, activated with anti-CD28 and anti-CD3for 3, 6, 12 h pooled (T103); T cell, TH0 clone Mot 72, anergic treatedwith specific peptide for 2, 7, 12 h pooled (T104); T cell, TH1 cloneHY06, resting (T107); T cell, TH1 clone HY06, activated with anti-CD28and anti-CD3 for 3, 6, 12 h pooled (T108); T cell, TH1 clone HY06,anergic treated with specific peptide for 2, 6, 12 h pooled (T109); Tcell, TH2 clone HY935, resting (T110); T cell, TH2 clone HY935,activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (T111); Tcells CD4+CD45RO− T cells polarized 27 days in anti-CD28, IL-1ζ, andanti IFN-γ, TH2 polarized, activated with anti-CD3 and anti-CD28 4 h(T116); T cell tumor lines Jurkat and Hut78, resting (T117); T cellclones, pooled AD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting(T118); T cell random γδ T cell clones, resting (T119); splenocytes,resting (B100); splenocytes, activated with anti-CD40 and IL-1ζ (B101);B cell EBV lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY, resting(B102); B cell line JY, activated with PMA and ionomycin for 1, 6 hpooled (B103); natural killer (NK) 20 clones pooled, resting (K100); NK20 clones pooled, activated with PMA and ionomycin for 6 h (K101); NKLclone, derived from peripheral blood of LGL leukemia patient, IL-2treated (K106); NK cytotoxic clone 640-A30-1, resting (K107);hematopoietic precursor line TF1, activated with PMA and ionomycin for1, 6 h pooled (C100); U937 premonocytic line, resting (M100); U937premonocytic line, activated with PMA and ionomycin for 1, 6 h pooled(M101); elutriated monocytes, activated with LPS, IFNγ, anti-IL-10 for1, 2, 6, 12, 24 h pooled (M102); elutriated monocytes, activated withLPS, IFNγ, IL-10 for 1, 2, 6, 12, 24 h pooled (M103); elutriatedmonocytes, activated with LPS, IFNγ, anti-IL-10 for 4, 16 h pooled(M106); elutriated monocytes, activated with LPS, IFNγ, IL-10 for 4, 16h pooled (M107); elutriated monocytes, activated LPS for 1 h (M108);elutriated monocytes, activated LPS for 6 h (M109); dendritic cells (DC)70% CD1a+, from CD34+ GM-CSF, TNFα 12 days, resting (D101); DC 70%CD1a+, from CD34+ GM-CSF, TNFα 12 days, activated with PMA and ionomycinfor 1 hr (D102); DC 70% CD1a+, from CD34+ GM-CSF, TNFα 12 days,activated with PMA and ionomycin for 6 hr (D103); DC 95% CD1a+, fromCD34+ GM-CSF, TNFα 12 days FACS sorted, activated with PMA and ionomycinfor 1, 6 h pooled (D104); DC 95% CD14+, from CD34+ GM-CSF, TNFα 12 daysFACS sorted, activated with PMA and ionomycin 1, 6 hr pooled (D105); DCCD1a+ CD86+, from CD34+ GM-CSF, TNFα 12 days FACS sorted, activated withPMA and ionomycin for 1, 6 h pooled (D106); DC from monocytes GM-CSF,IL-4 5 days, resting (D107); DC from monocytes GM-CSF, IL-4 5 days,resting (D108); DC from monocytes GM-CSF, IL-4 5 days, activated LPS 4,16 h pooled (D109); DC from monocytes GM-CSF, IL-4 5 days, activatedTNFα, monocyte supe for 4, 16 h pooled (D110); leiomyoma L11 benigntumor (X101); normal myometrium M5 (O115); malignant leiomyosarcoma GS1(X103); lung fibroblast sarcoma line MRC5, activated with PMA andionomycin for 1, 6 h pooled (C101); kidney epithelial carcinoma cellline CHA, activated with PMA and ionomycin for 1, 6 h pooled (C102);kidney fetal 28 wk male (O100); lung fetal 28 wk male (O101); liverfetal 28 wk male (O102); heart fetal 28 wk male (O103); brain fetal 28wk male (O104); gallbladder fetal 28 wk male (O106); small intestinefetal 28 wk male (O107); adipose tissue fetal 28 wk male (O108); ovaryfetal 25 wk female (O109); uterus fetal 25 wk female (O110); testesfetal 28 wk male (O111); spleen fetal 28 wk male (O112); adult placenta28 wk (O113); and tonsil inflamed, from 12 year old (X100).

Quantitative PCR using IL-1ξ specific primers on cDNA libraries showedlow expression levels across libraries. No significant signal abovebackground was detected, except for low (+), medium (++), or high (+++)relative signals were detected across libraries: mononuclear cells,activated; T cell, TH0 clone Mot 72, resting; T cell, TH0 clone Mot 72,activated; T cell, TH0 clone Mot 72, anergic; T cell, TH1 clone HY06,resting (++); T cell, TH1 clone HY06, activated (++); T cell, TH1 cloneHY06, anergic; T cell, TH2 clone HY935, resting; T cell, TH2 cloneHY935, activate”; T cells CD4+, TH2 polarized, activated; T cell linesJurkat and Hut78, resting; T cell clones, pooled, resting; T cell γδclones, resting, (+); CD28− T cell clone in pME (+); TR1 (Treg1) T cellclone (++); splenocytes, resting; splenocytes, activated; B cell EBVlines, resting; B cell line JY, activated; NK 20 clones pooled, resting(+); NK 20 clones pooled, activated; NK cytotoxic clone, resting (+++);hematopoietic precursor line TF1, activated; U937 premonocytic line,resting; U937 premonocytic line, activated; monocytes, LPS, IFNγ,anti-IL-10; monocytes, LPS, IFNγ, IL-10; monocytes, LPS, IFNγ,anti-IL-10, 4+16 h (+); monocytes, LPS, IFNγ, IL-10, 4+16 h; monocytes,LPS, 1 h; monocytes, LPS, 6 h; DC 70% CD1a+, from CD34+ GM-CSF, TNFα,resting; DC 70% CD1a+, from CD34+ GM-CSF, TNFα, activated 1 h; DC 70%CD1a+, from CD34+ GM-CSF, TNFα, activated 6 h; DC 95% CD1a+, from CD34+GM-CSF, TNFαt, activated 1+6 h; DC 95% CD14+, from CD34+ GM-CSF, TNFα,activated 1+6 h; DC CD1a+ CD86+, from CD34+ GM-CSF, TNFα, activated 1+6h; DC ex monocytes GM-CSF, IL-1ζ, resting; DC from monocytes GM-CSF,IL-1ζ, resting; DC from monocytes GM-CSF, IL-1ζ, LPS activated 4+16 h;DC from monocytes GM-CSF, IL-1ζ, monokine activated 4+16 h; epithelialcells, unstimulated; epithelial cells, IL-1β activated; lung fibroblastsarcoma line MRC5, activated; kidney epithelial carcinoma cell line CHA,activated; normal w.t. monkey lung; Ascaris-challenged monkey lung, 4 h;Ascaris-challenged monkey lung, 24 h; pool of two normal human lungsamples; pool of three heavy smoker human lung samples; allergic lungsample; Pneumocystic carnii pneumonia lung sample; normal w.t. monkeycolon; normal human colon; ulcerative colitis human colon sample; normalhuman thyroid; Hashimoto's thyroiditis thyroid sample; pool ofrheumatoid arthritis samples, human; normal human skin (+++); psoriasispatient skin sample (+++); tonsil inflamed; kidney fetal (++); lungfetal (+); liver fetal; heart fetal; brain fetal; gallbladder fetal (+);small intestine fetal (+); adipose tissue fetal; ovary fetal (+); uterusfetal; testes fetal; spleen fetal; placenta 28 wk (+); T cell, TH0 cloneMot 72, resting; T cell, TH0 clone Mot 72, activated; T cell, TH0 cloneB21, resting; T cell, TH0 clone B21, activated; T cell, TH1 cloneTA20-23, resting; T cell, TH1 clone TA20-23, activated; Jurkat cellline; and genomic DNA (++). Notably, expression was detected in normalskin, which is upregulated in psoriatic skin.

Because of the elevated signal in skin, various skin derived cellsamples were evaluated by quantitative PCR. While all of the signalswere, in absolute terms, quite low, no significant signal abovebackground was detected, except for low (+) or medium (++) levels in:fibroblasts IL-10 18 h (+); fibroblasts IL-10 6 h; fibroblasts IL-4 18h; fibroblasts IL-4 6 h; fibroblasts IFNγ 18 h; fibroblasts IFNγ 6 h;fibroblasts 3d pass untreated (+); keratinocytes IL-10 18 h (++);keratinocytes IL-10 6 h (++); keratinocytes IL-4 18 h (++);keratinocytes IL-4 6 h (++); keratinocytes IFNγ 18 h (++); keratinocytesIFNγ 6 h (++); keratinocytes 3d pass untreated (++); CLA+ T cells; CLA−T cells; Langerhans cells (+); fibroblasts 7th passage (+); andkeratinocytes 8th passage (+). These Langerhans and T cells wereisolated from skin biopsies. In summary, the IL-1ξ message is mainlyexpressed by the keratinocytes, and at lower levels in fibroblasts andLangerhans cells, with no significant expression in these T cells. Theexpression levels are comparable to that of the other IL-1 relatedmessages IL-1δ and IL-1ε, each at some 10-100 fold lower than levels ofexpression of primate IL-1α, IL-1β, or IL-1γ.

Likewise, in dendritic cells (DC), the signals were, in absolute terms,quite low. Dendritic cells (DC) are antigen-processing or presentingcells, and are found in all tissues of the body. They can be classifiedinto various categories, including: interstitial dendritic cells of theheart, kidney, gut, and lung; Langerhans cells in the skin and mucousmembranes; interdigitating dendritic cells in the thymic medula andsecondary lymphoid tissue; and blood and lymph dendritic cells. For areview of dendritic cells, see Steinman (1991) Annual Review ofImmunology 9:271-296; and Banchereau and Schmitt (eds. 1994) DendriticCells in Fundamental and Clinical Immunology Plenum Press, NY. Althoughdendritic cells in each of these compartments are CD45+ leukocytes thatapparently arise from bone marrow, they may exhibit differences thatrelate to maturation state and microenvironment.

These dendritic cells again did not express large amounts of message. Nosignificant signal above background was detected in pDC2 subset ofdendritic cells (monocyte derived DC). Low message levels were detectedin the DC1 (lymphocyte derived DC) day 0 and day 1 cultures. Higherlevels were detected in the day 2, 3, 5, 6, 7, and 8 DC1 cultures.

Using the information described herein for cloning species variants,expression of rodent IL-1ζ can be determined as above using, e.g., amurine homologue as for a detectable probe.

IV. Cloning of Species Counterparts of IL-1ζ

Various strategies are used to obtain species counterparts of primateIL-1ζ. One method is by cross hybridization using closely relatedspecies DNA probes. The degree of identity between mouse and human IL-1counterparts typically is as high as 70%. It may be useful to go intoevolutionarily similar species as intermediate steps. Another method isby using specific PCR primers based on the identification of blocks ofsimilarity between human and mouse IL-1 counterparts, e.g., areas ofhighly conserved polypeptide sequence.

In addition, the IL-1α, IL-1β, and IL-1RA genes cluster on the samehuman chromosome. The fourth known members of the IL-1 family, IL-1γ,which is most closely related to IL-1β, has been mapped to a differenthuman chromosome. Duplication of the intact IL-1α, IL-1β, IL-1RA genecluster, a potential genetic event explaining a proliferation ofadditional family members, would suggest the existence of two as yetunidentified IL-1 genes at the location of the IL-1γ locus. IL-1ζ is apotential candidate, and sequencing of the human IL-1γ locus may welllead to identification of the novel IL-1 genes.

V. Production of Mammalian IL-1ζ Protein

An appropriate, e.g., GST, fusion construct is engineered forexpression, e.g., in E. coli. For example, a mouse IGIF pGex plasmid isconstructed and transformed into E. coli. Freshly transformed cells aregrown in LB medium containing 50 μg/ml ampicillin and induced with IPTG(Sigma, St. Louis, Mo.). After overnight induction, the bacteria areharvested and the pellets containing IL-1ζ are isolated. The pellets arehomogenized in TE buffer (50 mM Tris-base pH 8.0, 10 mM EDTA and 2 mMpefabloc) in 2 liters. This material is passed through a microfluidizer(Microfluidics, Newton, Mass.) three times. The fluidized supernatant isspun down on a Sorvall GS-3 rotor for 1 h at 13,000 rpm. The resultingsupernatant containing the IL-1ζ is filtered and passed over aglutathione-SEPHAROSE column equilibrated in 50 mM Tris-base pH 8.0. Thefractions containing the IL-1ζ -GST fusion protein are pooled andcleaved with thrombin (Enzyme Research Laboratories, Inc., South Bend,Ind.). The cleaved pool is then passed over a Q-SEPHAROSE columnequilibrated in 50 mM Tris-base. Fractions containing IL-1ζ are pooledand diluted in cold distilled H₂O, to lower the conductivity, and passedback over a fresh Q-SEPHAROSE column. Fractions containing IL-1ζ arepooled, aliquoted, and stored in the −70° C. freezer.

Alternatively, the polypeptide may be produced in a non-fusion constructand expressed. The protein may be purified, either from secreted form,or from inclusion bodies, as appropriate.

Comparison of the CD spectrum with other IL-1 family members may suggestthat the protein is correctly folded. See Hazuda, et al. (1969) J. Biol.Chem. 264:1689-1693.

Human IL-1ζ epitope tagged protein gets secreted, e.g., from transientlytransfected 293 T cells, as shown by presence of C-terminally taggedprotein in the culture supernatant.

VI. Biological Assays with IL-1ζ

Biological assays confirmed IFN-γ inducing activity by IL-1γ on T cells.IL-1γ stimulates production of IFN-γ by purified NK cells, and thatinduction is strongly synergized with IL-12 or IL-2. Similar biologicalactivity should be exhibited by IL-1ζ or their antagonists.

The family of interleukins 1 contains molecules, each of which is animportant mediator of inflammatory disease. For a comprehensive review,see Dinarello (1996) “Biologic basis for interleukin-1 in disease” Blood87:2095-2147. There are indications that the various IL-1's playimportant roles in the initiation of disease, including the recentlyidentified IGIF/IL-1γ (e.g., Rothe, et al. (1997) “Active stage ofautoimmune diabetes is associated with the expression of a novelcytokine, IGIF, which is located near Idd2.” J. Clin. Invest.99:469-474. The finding of novel proteins related to the IL-1 familyfurthers the identification of molecules that provide the molecularbasis for initiation of disease and allow for the development oftherapeutic strategies of increased range and efficacy.

Similar biological assays as applied to other known members of thefamily should be performed with purified IL-1ζ.

VII. Preparation of Antibodies Specific for IL-1ζ

Inbred Balb/c mice are immunized intraperitoneally with recombinantforms of the protein, e.g., purified soluble IL-1ζ-FLAG or stabletransfected NIH-3T3 cells. Animals are boosted at appropriate timepoints with protein, with or without additional adjuvant, to furtherstimulate antibody production. Serum is collected, or hybridomasproduced with harvested spleens.

Alternatively, Balb/c mice are immunized with cells transformed with thegene or fragments thereof, either endogenous or exogenous cells, or withisolated membranes enriched for expression of the antigen. Serum iscollected at the appropriate time, typically after numerous furtheradministrations. Various gene therapy techniques may be useful, e.g., inproducing protein in situ, for generating an immune response. Operableassociation of heterologous promoters with natural gene sequences isalso provided, as are vectors encoding the DIRS3 with a receptorpartner. See, e.g., Treco, et al. WO96/29411 or U.S. Ser. No.08/406,030.

Monoclonal antibodies may be made. For example, splenocytes are fusedwith an appropriate fusion partner and hybridomas are selected in growthmedium by standard procedures. Hybridoma supernatants are screened forthe presence of antibodies which bind to the desired IL-1ζ, e.g., byELISA or other assay. Antibodies which specifically recognize IL-1ζ mayalso be selected or prepared, e.g., by immunoselection or relatedmethods.

In another method, synthetic peptides or purified protein are presentedto an immune system to generate monoclonal or polyclonal antibodies.See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene;and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press. In appropriate situations, the binding reagent is eitherlabeled as described above, e.g., fluorescence or otherwise, orimmobilized to a substrate for panning methods. Nucleic acids may alsobe introduced into cells in an animal to produce the antigen, whichserves to elicit an immune response. See, e.g., Wang, et al. (1993)Proc. Nat'l. Acad. Sci. 90:4156-4160; Barry, et al. (1994) BioTechniques16:616-619; and Xiang, et al. (1995) Immunity 2: 129-135.

VIII. Production of Fusion Proteins with IL-1ζ

Various fusion constructs are made with IL-1ζ. This portion of the geneis fused to an epitope tag, e.g., a FLAG tag, or to a two hybrid systemconstruct. See, e.g., Fields and Song (1989) Nature 340:245-246.

The epitope tag may be used in an expression cloning procedure withdetection with anti-FLAG antibodies to detect a binding partner, e.g.,receptor for the IL-1ζ. The two hybrid system may also be used toisolate proteins which specifically bind to IL-1ζ.

IX. Chromosome Mapping of IL-1ζ

Chromosome spreads are prepared. In situ hybridization is performed onchromosome preparations obtained from phytohemagglutinin-stimulatedlymphocytes cultured for 72 h. 5-bromodeoxyuridine is added for thefinal seven hours of culture (60 μg/ml of medium), to ensure aposthybridization chromosomal banding of good quality.

An appropriate fragment, e.g., a PCR fragment, is amplified with thehelp of primers on total B cell cDNA template, and cloned into anappropriate vector. The vector is labeled by nick-translation with ³H.The radiolabeled probe is hybridized to metaphase spreads, e.g., asdescribed in Mattel, et al. (1985) Hum. Genet. 69:327-331.

After coating with nuclear track emulsion (KODAK NTB₂), slides areexposed, e.g., for 18 days at 4° C. To avoid slipping of silver grainsduring the banding procedure, chromosome spreads are first stained withbuffered Giemsa solution and metaphase photographed. R-banding is thenperformed by the fluorochrome-photolysis-Giemsa (FPG) method andmetaphases rephotographed before analysis.

X. Structure Activity Relationship

Information on the criticality of particular residues is determinedusing standard procedures and analysis. Standard mutagenesis analysis isperformed, e.g., by generating many different variants at determinedpositions, e.g., at the positions identified above, and evaluatingbiological activities of the variants. This may be performed to theextent of determining positions which modify activity, or to focus onspecific positions to determine the residues which can be substituted toeither retain, block, or modulate biological activity.

Alternatively, analysis of natural variants can indicate what positionstolerate natural mutations. This may result from populational analysisof variation among individuals, or across strains or species. Samplesfrom selected individuals are analyzed, e.g., by PCR analysis andsequencing. This allows evaluation of population polymorphisms.

XI. Isolation of a Receptor for IL-1ζ

An IL-1ζ can be used as a specific binding reagent to identify itsbinding partner, by taking advantage of its specificity of binding, muchlike an antibody would be used. A binding reagent is either labeled asdescribed above, e.g., fluorescence or otherwise, or immobilized to asubstrate for panning methods.

The binding composition is used to screen an expression library madefrom a cell line which expresses a binding partner, i.e., receptor.Standard staining techniques are used to detect or sort intracellular orsurface expressed receptor, or surface expressing transformed cells arescreened by panning. Screening of intracellular expression is performedby various staining or immunofluorescence procedures. See also McMahan,et al. (1991) EMBO J. 10:2821-2832.

For example, on day 0, precoat 2-chamber permanox slides with 1 ml perchamber of fibronectin, 10 ng/ml in PBS, for 30 min. at roomtemperature. Rinse once with PBS. Then plate COS cells at 2-3×10⁵ cellsper chamber in 1.5 ml of growth media. Incubate overnight at 37° C.

On day 1 for each sample, prepare 0.5 ml of a solution of 66 μg/mlDEAE-dextran, 66 μm chloroquine, and 4 μg DNA in serum free DME. Foreach set, a positive control is prepared, e.g., of IL-1γ-FLAG cDNA at 1and 1/200 dilution, and a negative mock. Rinse cells with serum freeDME. Add the DNA solution and incubate 5 hr at 37° C. Remove the mediumand add 0.5 ml 10% DMSO in DME for 2.5 min. Remove and wash once withDME. Add 1.5 ml growth medium and incubate overnight.

On day 2, change the medium. On days 3 or 4, the cells are fixed andstained. Rinse the cells twice with Hank's Buffered Saline Solution(HBSS) and fix in 4% paraformaldehyde (PFA)/glucose for 5 min. Wash 3×with HBSS. The slides may be stored at −80° C. after all liquid isremoved. For each chamber, 0.5 ml incubations are performed as follows.Add HBSS/saponin (0.1%) with 32 μl/ml of 1 M NaN₃ for 20 min. Cells arethen washed with HBSS/saponin 1×. Add appropriate IL-1δ orIL-1δ/antibody complex to cells and incubate for 30 min. Wash cellstwice with HBSS/saponin. If appropriate, add first antibody for 30 min.Add second antibody, e.g., Vector anti-mouse antibody, at 1/200dilution, and incubate for 30 min. Prepare ELISA solution, e.g., VectorElite ABC horseradish peroxidase solution, and preincubate for 30 min.Use, e.g., 1 drop of solution A (avidin) and 1 drop solution B (biotin)per 2.5 ml HBSS/saponin. Wash cells twice with HBSS/saponin. Add ABC HRPsolution and incubate for 30 min. Wash cells twice with HBSS, secondwash for 2 min, which closes cells. Then add Vector diaminobenzoic acid(DAB) for 5 to 10 min. Use 2 drops of buffer plus 4 drops DAB plus 2drops of H₂O₂ per 5 ml of glass distilled water. Carefully removechamber and rinse slide in water. Air dry for a few minutes, then add 1drop of Crystal Mount and a cover slip. Bake for 5 min at 85-90° C.

Evaluate positive staining of pools and progressively subclone toisolation of single genes responsible for the binding.

Alternatively, IL-1ζ reagents are used to affinity purify or sort outcells expressing a receptor. See, e.g., Sambrook, et al. or Ausubel, etal.

Another strategy is to screen for a membrane bound receptor by panning.The receptor cDNA is constructed as described above. The ligand can beimmobilized and used to immobilize expressing cells. Immobilization maybe achieved by use of appropriate antibodies which recognize, e.g., aFLAG sequence of a IL-1ζ fusion construct, or by use of antibodiesraised against the first antibodies. Recursive cycles of selection andamplification lead to enrichment of appropriate clones and eventualisolation of receptor expressing clones.

Phage expression libraries can be screened by mammalian IL-1ζ.Appropriate label techniques, e.g., anti-FLAG antibodies, will allowspecific labeling of appropriate clones.

All citations herein are incorporated herein by reference to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by referenceincluding all figures and drawings.

Many modifications and variations of this invention, as will be apparentto one of ordinary skill in the art can be made to adapt to a particularsituation, material, composition of matter, process, process step orsteps, to preserve the objective, spirit and scope of the invention. Allsuch modifications are intended to be within the scope of the claimsappended hereto without departing from the spirit and scope of theinvention. The specific embodiments described herein are offered by wayof example only, and the invention is to be limited by the terms of theappended claims, along with the full scope of equivalents to which suchclaims are entitled; and the invention is not to be limited by thespecific embodiments that have been presented herein by way of example.

1. An isolated or recombinant polypeptide that: a) specifically bindspolyclonal antibodies generated against at least a 12 consecutive aminoacid segment of SEQ ID NO: 2 or 4; and b) comprises at least onesequence selected from: i) GENSGVK; EDWEKD; CCLEDPA; FVHTSR; KKFSIHD;VLVLDS; NLIAVP; FFALAS; SSASAEK; SLILLGV; FCLYCDK; PSLQLK; KLMKLAAQ;FIFYRAQ; SRNMLES; WFICTS; EPVGVT; or FSFQPVC (see SEQ ID NO: 2); or ii)FVHTSP; SPILLGV; or SWNMLES (see SEQ ID NO: 4).
 2. The polypeptide ofclaim 1: a) wherein said polypeptide comprises a plurality of saidsequences selected from said groups in section b); b) which specificallybinds to polyclonal antibodies generated against an immunogen selectedfrom SEQ ID NO: 2 or 4; or c) wherein said 12 consecutive amino acidsegment is selected from: GVKMGSEDWEKD; AGSPLEPGPSLP; SRKVKSLNPKKF;HDQDHKVLVLDS; NLIAVPDKNYIR; FALASSLSSASA; GQSHPSLQLKKE; MKLAAQKESARR;FYRAQVGSRNML; TSCNCNEPVGVT; FENRKHIEFSFQ; or PVCKAEMSPSEV (see SEQ IDNO: 2); or AVSPLEPGPSLP; SPKVKNLNPKKF; or FYRAQVGSWNML (see SEQ ID NO:4).
 3. The polypeptide of claim 2, wherein said polypeptide: i)comprises a mature protein; ii) lacks a post-translational modification;iii) is from a primate, including a human; iv) is a natural allelicvariant of IL-1ζ; v) has a length at least about 30 amino acids; vi)exhibits at least two non-overlapping epitopes that are specific for aprimate IL-1ζ; vii) exhibits a sequence identity over a length of atleast about 20 amino acids to SEQ ID NO: 2 or 4; viii) is notglycosylated; ix) has a molecular weight of at least 10 kD with naturalglycosylation; x) is a synthetic polypeptide; xi) is attached to a solidsubstrate; xii) is conjugated to another chemical moiety; xiii) is a5-fold or less substitution from natural sequence; or xiv) is a deletionor insertion variant from a natural sequence.
 4. A composition of mattercomprising: a) a sterile polypeptide of claim 2; b) said sterilepolypeptide of claim 2 and a carrier, wherein said carrier is: i) anaqueous compound, including water, saline, and/or buffer; and/or ii)formulated for oral, rectal, nasal, topical, or parenteraladministration.
 5. A fusion protein having a polypeptide sequence ofclaim 2 further comprising: a) mature polypeptide of claim 2; b) adetection or purification tag, including a FLAG, His 6, or Ig sequence;or c) sequence of another cytokine or chemokine.
 6. A kit comprising apolypeptide of claim 2, and: a) a compartment comprising saidpolypeptide; and/or b) instructions for use or disposal of reagents insaid kit.
 7. A binding compound comprising an antigen binding site froman antibody, which specifically binds to a mature polypeptide of claim2, wherein: a) said mature polypeptide is a primate IL-1ζ; b) saidbinding compound is an Fv, Fab, or Fab2 fragment; c) said bindingcompound is conjugated to another chemical moiety; or d) said antibody:i) is raised against a 12 consecutive amino acid segment of SEQ ID NO: 2or 4; ii) is raised against a mature IL-1ζ; iii) is raised to a purifiedprimate IL-1ζ; iv) is immunoselected; v) is a polyclonal antibody; vi)binds to a denatured IL-1ζ; vii) exhibits a Kd to antigen of at least 30μM; viii) is attached to a solid substrate, including a bead or plasticmembrane; ix) is in a sterile composition; or x) is detectably labeled,including a radioactive or fluorescent label.
 8. A kit comprising saidbinding compound of claim 7, and: a) a compartment comprising saidbinding compound; and/or b) instructions for use or disposal of reagentsin said kit.
 9. A composition comprising: a) sterile binding compound ofclaim 7, or b) said binding compound of claim 7 and a carrier, whereinsaid carrier is: i) an aqueous compound, including water, saline, and/orbuffer; and/or ii) formulated for oral, rectal, nasal, topical, orparenteral administration.
 10. An isolated or recombinant nucleic acidencoding a polypeptide of claim 2, wherein: a) said polypeptide of claim2 is a primate IL-1ζ; or b) said nucleic acid: i) encodes an antigenicpeptide sequence of SEQ ID NO: 2 or 4; ii) encodes a plurality ofantigenic peptide sequences of SEQ ID NO: 2 or 4; iii) exhibits at leastabout 80% identity to a natural cDNA encoding said segment; iv) is anexpression vector; v) further comprises an origin of replication; vi) isfrom a natural source; vii) comprises a detectable label; viii)comprises synthetic nucleotide sequence; ix) is less than 6 kb,preferably less than 3 kb; x) is from a rodent; xi) comprises a naturalfull length coding sequence; xii) is a hybridization probe for a geneencoding said IL-1ζ; or xiii) is a PCR primer, PCR product, ormutagenesis primer; or xiv) encodes an IL-1ζ polypeptide.
 11. A cell,transformed with said nucleic acid of claim
 10. 12. The cell of claim11, wherein said cell is: a) a prokaryotic cell; b) a eukaryotic cell;c) a bacterial cell; d) a yeast cell; e) an insect cell; f) a mammaliancell; g) a mouse cell; h) a primate cell; or i) a human cell.
 13. A kitcomprising said nucleic acid of claim 10, and: a) a compartment furthercomprising a primate IL-1ζ polypeptide; and/or b) instructions for useor disposal of reagents in said kit.
 14. An isolated or recombinantnucleic acid that hybridizes under wash conditions of 30° C. and lessthan 2M salt to SEQ ID NO:
 1. 15. The nucleic acid of claim 14, wherein:a) said wash condition is at 45° C. and/or 500 mM salt; or b) saidnucleic acid encodes at least 12 contiguous amino acids of SEQ ID NO: 2or
 4. 16. The nucleic acid of claim 15, wherein: a) said wash conditionis at 55° C. and/or 150 mM salt; or b) said nucleic acid encodes atleast 17 contiguous amino acids of SEQ ID NO: 2 or
 4. 17. A method ofmodulating a cell involved in an inflammatory response comprisingcontacting said cell with an agonist or antagonist of a primate IL-1ζpolypeptide of claim
 1. 18. The method of claim 17, wherein: a) saidcontacting is in combination with an agonist or antagonist of IL-1α,IL-1RA, IL-1β, IL-1γ, IL-1δ, IL-1ε, IL-2, and/or IL-12; b) saidcontacting is with an antagonist, including binding compositioncomprising an antibody binding site which specifically binds an IL-1ζ;or c) said modulating is regulation of IFN-γ production.
 19. A bindingcompound comprising an antigen binding portion from an antibody, whichspecifically binds to a primate protein of claim 1, wherein: a) saidprotein is a human protein; b) said binding compound is an Fv, Fab, orFab2 fragment; c) said binding compound is conjugated to anotherchemical moiety; or d) said antibody: i) is raised against a polypeptidesequence of a mature polypeptide comprising at least 12 consecutiveamino acids of SEQ ID NO: 2 or 4; ii) is raised against a mature primateIL-1ζ; iii) is raised to a purified primate IL-1ζ; iv) isimmunoselected; v) is a polyclonal antibody; vi) binds to a denaturedprimate IL-1ζ; vii) exhibits a Kd to antigen of at least 30 μM; viii) isattached to a solid substrate, including a bead or plastic membrane; ix)is in a sterile composition; or x) is detectably labeled, including aradioactive or fluorescent label.
 20. A method of: A) making an antibodyof claim 19, comprising immunizing an immune system with an immunogenicamount of: a) a primate IL-1ζ polypeptide; or b) a peptide sequencecomprising at least 12 consecutive amino acids of SEQ ID NO: 2 or 4; thereby causing said antibody to be produced; or B) producing anantigen:antibody complex, comprising contacting a primate IL-1ζpolypeptide with an antibody of claim 19 thereby allowing said complexto form.